U.S. patent application number 13/965092 was filed with the patent office on 2014-01-09 for antibodies against influenza virus and methods of use thereof.
This patent application is currently assigned to BURNHAM INSTITUTE FOR MEDICAL RESEARCH. The applicant listed for this patent is Burnham Institute for Medical Research, Dana-Farber Cancer Institute, Inc.. Invention is credited to Robert C. Liddington, Wayne A. Marasco, Jianhua Sui.
Application Number | 20140011982 13/965092 |
Document ID | / |
Family ID | 40796093 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011982 |
Kind Code |
A1 |
Marasco; Wayne A. ; et
al. |
January 9, 2014 |
Antibodies Against Influenza Virus and Methods of Use Thereof
Abstract
The invention provides human scFv antibodies and monoclonal
antibodies that neutralize influenza virus. Also provided are
methods of treating and/or preventing a influenza related disease
or disorder such bird flu The invention also provides methods of
vaccinating a patient against influenza. Also provided are methods
of diagnosing influenza-related diseases or disorders and methods
of detecting the presence of a influenza in a sample.
Inventors: |
Marasco; Wayne A.;
(Wellesley, MA) ; Sui; Jianhua; (Waltham, MA)
; Liddington; Robert C.; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Burnham Institute for Medical Research
Dana-Farber Cancer Institute, Inc. |
La Jolla
Boston |
CA
MA |
US
US |
|
|
Assignee: |
BURNHAM INSTITUTE FOR MEDICAL
RESEARCH
92037
CA
DANA-FARBER CANCER INSTITUTE, INC.
Boston
MA
|
Family ID: |
40796093 |
Appl. No.: |
13/965092 |
Filed: |
August 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12746622 |
Oct 4, 2010 |
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PCT/US2008/085876 |
Dec 8, 2008 |
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13965092 |
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61005725 |
Dec 6, 2007 |
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61091599 |
Aug 25, 2008 |
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Current U.S.
Class: |
530/387.3 ;
530/388.15; 536/23.53 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 2760/16122 20130101; C07K 2319/40 20130101; C07K 2317/565
20130101; C07K 2317/622 20130101; C07K 2317/92 20130101; C07K
2317/76 20130101; C07K 16/1018 20130101; C07K 2317/21 20130101;
C07K 2317/52 20130101; A61P 31/16 20180101; A61K 2039/505 20130101;
C07K 14/005 20130101 |
Class at
Publication: |
530/387.3 ;
530/388.15; 536/23.53 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Goverment Interests
GRANT SUPPORT
[0002] This invention was made with United States Government
support under National Institutes of Health Grants U01 AI074518-01.
The United States Government has certain rights in the invention.
Claims
1. An isolated monoclonal antibody, or scFv fragment thereof,
wherein said monoclonal antibody binds to an epitope in the stem
region of the hemagglutinin (HA) protein of an influenza virus, and
neutralizes the influenza A virus, and wherein the epitope
comprises the amino acids at positions 18, 38, 40, and 291 of the
influenza A virus HA1 polypeptide.
2. The monoclonal antibody, or scFv fragment thereof, of claim 1,
wherein the epitope further comprises the amino acids at position
18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53 and 56 of the influenza
A virus HA2 polypeptide.
3. The monoclonal antibody, or scFv fragment thereof, of claim 1,
wherein said monoclonal antibody competes with the binding of
monoclonal antibody F10 to the HA protein.
4. The monoclonal antibody, or scFv fragment thereof, of claim 1,
wherein said monoclonal antibody is a fully human antibody.
5. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said monoclonal antibody, or scFv fragment
thereof, stabilizes the neutral pH conformation of the HA
protein.
6. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said influenza A virus is an H1a cluster influenza
virus or an H1b cluster influenza virus.
7. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said influenza A virus is an H1, H2, H5, H6, or
H11 subtype.
8. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said monoclonal antibody, or scFv fragment
thereof, inhibits viral and cell membrane fusion.
9. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said isolated monoclonal antibody, or scFv
fragment thereof, binds to HA with a Kd value of 1 pm-200 mM.
10. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 1, wherein said monoclonal antibody, or scFv fragment
thereof, binds to HA with a Kd value of about 100-200 pM.
11. An isolated monoclonal antibody, or scFv fragment thereof,
wherein the monoclonal antibody or scFv fragment thereof, binds to
the stem region of the HA protein of an influenza A virus and
comprises (a) a heavy chain with a CDR1 comprising amino acid
sequence EVTFSSFA (SEQ ID NO: 120); (b) a heavy chain with a CDR2
comprising amino acid sequence ISPMFGTP (SEQ ID NO: 128); (c) a
heavy chain with a CDR3 comprising amino acid sequence
ARSPSYICSGGTCVFDH (SEQ ID NO: 134); (d) a light chain with a CDR1
comprising aft amino acid sequence SNNVGNQG (SEQ ID NO: 142); (e) a
light chain with a CDR2 comprising a amino acid sequence RNN (SEQ
ID NO: 152); and (f) a light chain with a CDR3 comprising a amino
acid sequence STWDSSLSAVV (SEQ ID NO: 162).
12. The isolated monoclonal antibody or scFv fragment thereof of
claim 11, wherein the monoclonal antibody, or scFv fragment
thereof, comprises a heavy chain variable domain comprising amino
acid sequence SEQ ID NO: 112, and a light chain variable domain
comprising amino acid sequence SEQ ID NO: 20.
13. The monoclonal antibody, or scFv fragment thereof, of claim 11,
wherein said monoclonal antibody is a fully human antibody.
14. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said monoclonal antibody, or scFv fragment
thereof, stabilizes the neutral pH conformation of the HA
protein.
15. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said influenza A virus is an H1a cluster
influenza virus or an H1b cluster influenza virus.
16. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said influenza A virus is an H1, H2, H5, H6, or
H11 subtype.
17. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said monoclonal antibody, or scFv fragment
thereof, inhibits viral and cell membrane fusion.
18. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said isolated monoclonal antibody, or scFv
fragment thereof, binds to HA with a Kd value of 1 pm-200 mM.
19. The isolated monoclonal antibody, or scFv fragment thereof, of
claim 11, wherein said monoclonal antibody, or scFv fragment
thereof, binds to HA with a Kd value of about 100-200 pM.
20. The isolated nucleic acid molecule encoding the isolated
monoclonal antibody, or scFv fragment thereof, of claim 11.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/746,622, filed Oct. 4, 2010, which is a National Stage Entry
Application and claims benefit of priority of International
Application No. PCT/US2008/085876, filed Dec. 8, 2008, which claims
benefit of priority under 35 USC .sctn.119(e) to U.S. Provisional
Application No. 61/005,725, filed Dec. 6, 2007 and U.S. Provisional
Application No. 61/091,599, filed Aug. 25, 2008, the contents of
which are incorporated herein by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0003] The contents of the text file named
"20363-049C01US_ST25.txt", which was created on Aug. 12, 2013 and
is 59 KB in size, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0004] This invention relates generally to anti-viral antibodies as
well as to methods for use thereof.
BACKGROUND
[0005] An influenza pandemic represents one of the greatest acute
infectious threats to human health. The 1918-1919 influenza
pandemic caused an estimated 500,000 deaths in the United States,
making it the most fatal event in all of US history. The spread of
highly pathogenic avian influenza (HPAI) H5N1 influenza across Asia
and now to the middle east and northern Africa creates a
substantial risk for a new pandemic to arise.
[0006] Natural variation as well as escape mutants suggests that
continued evolution of the virus should impact the decision on
which strain(s) should be used for passive and active immunization.
Although a number of important epitope mapping and neutralization
escape studies have been reported new neutralizing antibodies and
related structural studies are needed to develop immunization
strategies against HPAI H5N1. The challenges to developing a
protective vaccine against HPAI H5N1 are formidable and new
approaches are needed to prevent and treat human infection by an
ever changing enemy. There is a need to rapidly develop therapeutic
strategies to elicit protective hosts immunity, both passively and
actively.
[0007] Tremendous advances in the field of human antibody (Ab)
engineering have been made. Monoclonal antibody (Mab) based
immunotherapies are now becoming standard of care in an increasing
number of human diseases including RSV. The shift toward de novo
human Mab isolation and their clinical use is in part due to new
antibody display and other library screening techniques that are
now be exploited to build human antibodies with high affinity and
specificity. Human Mab immunotherapies can provide an increasingly
important role in clinical management of human diseases.
SUMMARY OF THE INVENTION
[0008] The invention is based upon the discovery of monoclonal
antibodies which neutralize the influenza virus, e.g. influenza A
virus. The influenza A virus is a Group I influenza A virus such as
a H1 cluster influenza virus. The H1 cluster influenza virus is an
H1a cluster or an H1b cluster. The monoclonal antibody is fully
human. In various aspects, the monoclonal antibody is a bivalent
antibody, a monovalent antibody, a single chain antibody or
fragment thereof. Specifically, such monoclonal bind to an epitope
on the stem region of the hemagglutinin protein (HA), such as HA1
or HA2 polypeptide. The epitope is non-linear.
[0009] Optionally, the epitope comprises both the HA-1 and HA-2.
The epitope is non-linear. In some embodiments the epitope
comprises the amino acid position 18, 38, 40, 291 of the HA1
polypeptide and the amino acid at position 18, 19, 20, 21, 38, 41,
42, 45, 49, 52, 53 and 56 of the HA2 polypeptide.
[0010] Exemplary monoclonal antibodies include monoclonal antibody
D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or an antibody
that binds to the same epitope as D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98.
[0011] The monoclonal antibodies of the invention can have the
binding affinity of monoclonal antibody D7, D8, F10, G17, H40, A66,
D80, E88, E90, or H98. Alternatively, the binding affinity can
range about 1 pM to about 200 mM. The monoclonal antibodies of the
invention function to inhibit viral and cell membrane fusion.
[0012] The monoclonal antibody has a heavy chain variable amino
acid sequence containing SEQ ID NOS: 2, 6, 12, 18, 24, 28, 32, and
36 and/or a light chain variable amino acid sequence containing SEQ
ID NOS: 4, 8, 14, 16, 20, 22, 26, 30, 34, and 38.
[0013] The monoclonal antibody, has a heavy chain variable nucleic
acid sequence containing SEQ ID NOS: 1, 5, 13, 15, 21, 23, 29, 33,
37, and 40 and or a light chain variable nucleic acid sequence
containing SEQ ID NOS: 3, 9, 11, 17, 19, 25, 27, 31, 35, 39, and
42
[0014] Also provided by the invention is a monoclonal
anti-influenza hemagglutinin protein antibody or fragment thereof,
where the antibody has a V.sub.H CDR1 region having the amino acid
sequence SYAFS (SEQ ID NO: 43), TNAFS (SEQ ID NO: 44), AYAFT (SEQ
ID NO: 45), SFAIS (SEQ ID NO: 46), SYAIS (SEQ ID NO: 47), GYYIH
(SEQ ID NO: 48), MTAFT (SEQ ID NO: 49), or DNAIS (SEQ ID NO: 50); a
V.sub.H CDR2 region having the amino acid sequence
GIIPMFGTPNYAQKFQG (SEQ ID NO: 51), GVIPLFRTASYAQNVQG (SEQ ID NO:
52), GIIGMFGTANYAQKFQG (SEQ ID NO: 53), GISPMFGTPNYAQKFQG (SEQ ID
NO: 54), GIIGVFGVPKYAQKFQG (SEQ ID NO: 55), WINPMTGGTNYAQKFQV (SEQ
ID NO: 56), GISPIFRTPKYAQKFQG (SEQ ID NO: 57), or GIIPIFGKPNYAQKFQG
(SEQ ID NO: 58); a V.sub.H CDR3 region having the amino acid
sequence SSGYYYG GGFDV (SEQ ID NO: 59), SSGYHFGRSHFDS (SEQ ID NO:
60), GLYYYESSLDY (SEQ ID NO: 61), SPSYICSGGTCVFDH (SEQ ID NO: 62),
EPGYYVGKNGFDV (SEQ ID NO: 63), GASVLRYFDWQPEALDI (SEQ ID NO: 64),
TLSSYQPNNDAFAI (SEQ ID NO: 65), or DSDAYYYGSGGMDV (SEQ ID NO: 66);
V.sub.L CDR1 region having amino acid sequence TGSSSNIGNYVA (SEQ ID
NO: 67), TGSSSNIAANYVQ (SEQ ID NO: 68), TGTSSDVGGYNSVS (SEQ ID NO:
69), TGNSNNVGNQGAA (SEQ ID NO: 70), TGDSNNVGHQGTA (SEQ ID NO: 71),
GGNNIGGYSVH (SEQ ID NO: 72), RASQSVSSYLA (SEQ ID NO: 73),
RASQSLSSKYLA (SEQ ID NO: 74), TGSSSNIGNYVA (SEQ ID NO: 75),
SGSSSNIGSNTVN (SEQ ID NO: 76), RASQSISSYLN (SEQ ID NO: 77), or
TLSSGHSNYIIA (SEQ ID NO: 78); a V.sub.L CDR2 region having the
amino acid sequence SNSDRPS (SEQ ID NO: 79), EDDRRPS (SEQ ID NO:
80), EVTKRPSU (SEQ ID NO: 81), RNNDRPS (SEQ ID NO: 82), RNGNRPS
(SEQ ID NO: 83), DDKDRPS (SEQ ID NO: 84), DASNRAT (SEQ ID NO: 85),
GASSRAT (SEQ ID NO: 86), SNNQRPS (SEQ ID NO: 87), AASSLQR (SEQ ID
NO: 88), SNEQRPS (SEQ ID NO: 89), or VNSDGSHTKGD (SEQ ID NO: 90)
and/or a V.sub.L CDR3 region having the amino acid sequence
QSYDSLSAYV (SEQ ID NO: 91), QSYDTNNHAV (SEQ ID NO: 92), CSYAGHSAYV
(SEQ ID NO: 93), STWDSSLSAVV (SEQ ID NO: 94), SVWDSSLSAWV (SEQ ID
NO: 95), QVWDSGNDRPL (SEQ ID NO: 96), QQYGSSPQV (SEQ ID NO: 97),
QQYDGVPRT (SEQ ID NO: 98), QSYDSRLSASL (SEQ ID NO: 99), QQYDSSPYT
(SEQ ID NO: 100), ASWDDNLSGWV (SEQ ID NO: 101), or ETWDTKIHV (SEQ
ID NO: 102).
[0015] In a further aspect, the invention provides An isolated an
monoclonal anti-influenza hemagglutinin protein antibody or
fragment thereof where the antibody has a VH amino acid sequence
encoded by the VH germline gene IGHV1-69*01 and the amino acid at
position: a) 27 is a valine; b) 28 is threonine; c) 30 is serine;
d) 31 is serine; e) 54 is methionine; f) 55 is phenylalanine; g) 58
is threonine; h) 100 is proloine; i) 101 is serine; j) 102 is
tyrosine; k) 103 is isoleucine and 105 is serine.
[0016] In another aspect, the invention provides a method of
preventing or treating a disease or disorder caused by an influenza
virus by administering to a person at risk of suffering from said
disease or disorder, a therapeutically effective amount of a
monoclonal antibody or scFV antibody described herein. The
monoclonal antibody or scFV antibody is administered at a dose
sufficient to neutralize the influenza virus. In embodiments of the
invention, the method also includes administering an anti-viral
drug, a viral entry inhibitor or a viral attachment inhibitor. The
anti-viral drug is neuraminidase inhibitor such as zanamivir, or
oseltamivir phosphate, a HA inhibitor, a sialic acid inhibitor or
an M2 ion channel such as amantadine or rimantadine. The antibody
is administered prior t or after exposure to an influenza virus
[0017] In another aspect, the invention provides a method of
detecting the presence of a an influenza virus in a sample by
contacting the sample with a monoclonal antibody as described
herein, and detecting the presence or absence of an
antibody-antigen complex, thereby detecting the presence of a
influenza virus in a sample. The test sample is generally obtained
from blood, hair, cheek scraping or swab, saliva, biopsy, urine,
feces, sputum, nasal aspiration, or semen.
[0018] The invention is further based upon the discovery of a
protocol for generating broadly neutralizing human antibodies that
target a highly conserved epitope in the stem region of HA. By
using the trimeric H5 ectodomain expressed in baculovirus which
produces shorter N-glycans and uncharged mannoses absorbed on a
plastic surface, allowed for the dominant presentation of the stem
epitope while masking the normally immunodominat globular head.
[0019] Accordingly, also included in the invention is a method of
producing an isolated antibody that specifically binds a pathogenic
enveloped virus by exposing a single chain or Fab expression
library to a membrane fusion protein of the virus, identifying an
antibody in the library that specifically binds said protein; and
isolating the antibody from the library. Preferably, the fusion
protein is immobilized on a solid surface, e.g. plastic. In various
aspects the fusion protein has modified glycosylations compared to
a wild type fusion protein. For example, the fusion is produced in
a non-mammalian cell, such as an insect cell. The fusion protein is
for example a trimeric hemagglutinin (HA) protein
[0020] The invention further provides a method of vaccinating a
subject against pathogenic enveloped virus such as an influenza
virus by administering to the subject a membrane fusion protein
(e.g., a trimeric hemagglutinin (HA) protein coated) or embedded in
a biologically compatible matrix. In various aspects the fusion
protein has modified glycosylations compared to a wild type fusion
protein.
[0021] In another aspect, the invention provides a composition
comprising a monoclonal antibody as described herein and kits
containing the composition in one or more containers and
instructions for use.
[0022] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and are not intended to be
limiting.
[0023] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is an illustration of the structure of the A/Vietnam
1203/04 trimer. The receptor binding site and antigenic variation
sites are highlighted on the monomer.
[0025] FIG. 1B is an illustration showing the location of amino
acid residues in the HA of H5N1 influenza viruses that are under
positive selection
[0026] FIG. 2 is a schematic illustration of convergent combination
Immunotherapy for H5N1.
[0027] FIG. 3 is an illustration showing an amino acid sequence
comparison of the framework regions 1-4 (FR1-4) and the
complementary-determining regions 1-3(CDR1-3) for both the VH
(full-length sequences disclosed as SEQ ID NOS: 110-117,
respectively, in order of appearance) and VL (full-length sequences
disclosed as SEQ ID NOS: 8, 14, 20, 26, 30, 34, 16, 38, 22, and 10,
respectively, in order of appearance) of the anti-influenza
antibodies of the invention. FR and CDR regions are defined using
Kabat database. The VH and VL gene name are shown on the right
(using IMGT database). Dots show sequence identity to the consensus
sequence. Hyphens represent gaps. FIG. 3 discloses the VH "CDR1" as
SEQ ID NOS: 118-125, "CDR2" as SEQ ID NOS: 126-133, and "CDR3" as
SEQ ID NOS: 170 and 134-139, all respectively, in order of
appearance. For example, antibody F10/E90 has a heavy chain
consisting of a CDR1 with the sequence EVTFSSFA (SEQ ID NO: 120); a
CDR2 with the sequence ISPMFGTP (SEQ ID NO: 128); and a CDR3 with
the sequence ARSPSYICSGGTCVFDH (SEQ ID NO: 134). Antibody D8/D80
has a heavy chain consisting of a CDR1 with the sequence GGTFSAYA
(SEQ ID NO: 121); a CDR2 with the sequence IIGMFGTA (SEQ ID NO:
129); and a CDR3 with the sequence ARGLYYYESSFDY (SEQ ID NO: 136).
Antibody A66/E88 has a heavy chain consisting of a CDR1 with the
sequence GGPFSMTA (SEQ ID NO: 122); a CDR2 with the sequence
ISPIFRTP (SEQ ID NO: 130); and a CDR3 with the sequence
ARTLSSYQPNNDAFAI (SEQ ID NO: 136). Antibody G17 has a heavy chain
consisting of a CDR1 with the sequence GVTFSSYA (SEQ ID NO: 123); a
CDR2 with the sequence IIGVFGVP (SEQ ID NO: 131); and a CDR3 with
the sequence AREPGYYVGKNGFDV (SEQ ID NO: 137). Antibody D7/H98 has
a heavy chain consisting of a CDR1 with the sequence GGIFNTNA (SEQ
ID NO: 124); a CDR2 with the sequence VIPLFRTA (SEQ ID NO: 132);
and a CDR3 with the sequence ARSSGYHFRSH (SEQ ID NO: 138). Antibody
H40 has a heavy chain consisting of a CDR1 with the sequence
GYTFTGYY (SEQ ID NO: 125); a CDR2 with the sequence INPMTGGTP (SEQ
ID NO: 133); and a CDR3 with the sequence ARGASVLRYFDWQPEALDI (SEQ
ID NO: 139). FIG. 3 also discloses the VL "CDR1" as SEQ ID NOS:
140-149, "CDR2" as SEQ ID NOS: 150-159; and "CDR3" as SEQ ID NOS
160-169, all respectively, in order of appearance. For example,
antibody D7 has a light chain consisting of a CDR1 with the
sequence SGNIAANY (SEQ ID NO: 140); a CDR2 with the sequence EDD
(SEQ ID NO: 150); and a CDR3 with the sequence QTYDTNNHAV (SEQ ID
NO: 160). Antibody D8 has a light chain consisting of a CDR1 with
the sequence SSDVGGYNS (SEQ ID NO: 141); a CDR2 with the sequence
EVT (SEQ ID NO: 151); and a CDR3 with the sequence CSYAGHSAYV (SEQ
ID NO: 161). Antibody F10 has a light chain consisting of a CDR1
with the sequence SNNVGNQG (SEQ ID NO: 142); a CDR2 with the
sequence RNN (SEQ ID NO: 152); and a CDR3 with the sequence
STWDSSLSAVV (SEQ ID NO: 162). Antibody G17 has a light chain
consisting of a CDR1 with the sequence SNNVGHQG (SEQ ID NO: 143); a
CDR2 with the sequence RNG (SEQ ID NO: 153); and a CDR3 with the
sequence SVWDSSLSAWV (SEQ ID NO: 163). Antibody H40 has a light
chain consisting of a CDR1 with the sequence NIGGYS (SEQ ID NO:
144); a CDR2 with the sequence DDK (SEQ ID NO: 154); and a CDR3
with the sequence QVWDSGNDRPL (SEQ ID NO: 164). Antibody A66 has a
light chain consisting of a CDR1 with the sequence QSVSSY (SEQ ID
NO: 145); a CDR2 with the sequence DAS (SEQ ID NO: 155); and a CDR3
with the sequence QQYGSSPQ (SEQ ID NO: 165). Antibody D80 has a
light chain consisting of a CDR1 with the sequence QSLSSKY (SEQ ID
NO: 146); a CDR2 with the sequence GAS (SEQ ID NO: 156); and a CDR3
with the sequence QQYDGVPRT (SEQ ID NO: 166). Antibody E88 has a
light chain consisting of a CDR1 with the sequence SSNIGSNT (SEQ ID
NO: 147); a CDR2 with the sequence SNN (SEQ ID NO: 157); and a CDR3
with the sequence QSYDSRLSASL (SEQ ID NO: 167). Antibody E90 has a
light chain consisting of a CDR1 with the sequence QSISSY (SEQ ID
NO: 148); a CDR2 with the sequence AAS (SEQ ID NO: 158); and a CDR3
with the sequence QQYDSSPYT (SEQ ID NO: 168). Antibody H98 has a
light chain consisting of a CDR1 with the sequence TSNIGRNH (SEQ ID
NO: 149); a CDR2 with the sequence SNE (SEQ ID NO: 159); and a CDR3
with the sequence ASWDDNLSGWV (SEQ ID NO: 169). FIG. 3 also
discloses "QVQLVQSGAEV" as SEQ ID NO: 171 and "VTVSS" as SEQ ID NO:
172.
[0028] FIG. 4 shows in vitro neutralization of anti-H5 antibodies.
(a) (Top panel) The 10 nAbs were converted to soluble scFv-Fcs
(single chain fragment of variable region linked with Hinge, CH2
and CH3) and evaluated for neutralizing activity against
H5-TH04-pseudotyped viruses. The percentage neutralization at 2
concentrations is shown with standard error bars. The mAb
80R.sup.16 was used as the control (Ctrl.). (Middle and bottom
panels), Neutralization of wild type H5-VN04 and H5-1N05 by the 10
scFv-Fcs at three concentrations using a plaque reduction assay.
Results are consistent with those obtained from a
microneutralization assay (data not shown). (b) Cross subtype
neutralization. nAbs D8, F10 and A66 all neutralized H5-TH04,
H1-SC1918 ((A/South Carolina/1/1918 (H1N1)), H1-PR34 (A/Puerto
Rico/8/34 (H1N1)), H2-JP57 (A/Japan/305/57(H2N2)) and H6-NY98
(A/chicken/New York/14677-13/1998(H6N2)) pseudotyped viruses. (c)
Microneutralization assay. Neutralization titers of F10 against
wild-type H5N1, H1N1 (A/Ohio/83 ("H1-OH83")) and H2N2 (A/Ann
Arbor/6/60 ("H2-AA60")) compared with a murine mAb 21G8.6 raised
against H5N1 and Ctrl. 80R (1 mg/mL Ab stock solution). "<"
indicates a titer of less than 20. Results represent two
independent experiments.
[0029] FIG. 5 shows the Neutralization mechanism. (a) Virus binding
inhibition assay using full-length HA of H5-TH04-pseudotyped HIV-1
viruses. The binding of virus to cells in Ab-treated groups were
compared with binding to cells in the absence of Ab (defined as
100%). None of the three nAbs (D8, F10 and A66) inhibited virus
binding to cells, while a mouse anti-H5 mAb, 17A2.1.2, and ferret
anti-H5N1 serum, both of which inhibit haemagglutination and
presumably bind to the head, reduced binding significantly. (b)
Inhibition of syncytia formation by nAbs. HeLa cells were
transfected with H5-TH04-expressing plasmid. Syncytia formation
induced by brief exposure to pH 5.0 buffer was completely inhibited
by nAbs D8, F10 and A66, at 20 .mu.g/ml, whereas control and an
anti-HA1 mAb (2A) at the same concentration had no effect.
[0030] FIGS. 6A-6D show the structure of the H5-F10 epitope, and
sequence/structural conservation. (a) Structure of the H5 trimer
bound to F10 (scFv). H5 is very similar to the uncomplexed
structure.sup.21 (pairwise RMSD (C.alpha.)=1.0 and 0.63 .ANG. for 2
independent trimers). HA1, HA2, the .alpha.A-helix of HA2, the
"fusion peptide" (FP), and F10 (VH and VL) are colour-coded. The
third F10 is hidden behind the stem. Note that VL makes no contacts
with H5. (b) Surface of the central stem region. One monomer is
coloured by chain; the path of FP through the epitope (red) is
outlined; mutations not affecting binding are in cyan. The fusion
peptide (FP and FP') is visible in two monomers. Epitope residues
are labeled white (HA2) or yellow (HA1). (c) Close-up of the
epitope showing the tip of F10 (red ribbon) and selected CDR
side-chains. Of 1500 .ANG..sup.2 buried surface at the interface,
43% involves hydrophobic interactions. (d) Overlay of HA subtypes:
H1, H5 and H9 (Group 1) in shades of red/yellow (1RU7, 2IBX and
1JSD); H3 and H7 (Group 2) in shades of blue (1MQL and 1TI8). RMSDs
for pairwise overlays are 0.56.+-.0.11 .ANG. (observed range, Group
1); 0.75 .ANG. (Group 2); and 1.21.+-.0.12 .ANG. between groups.
Consistent differences between groups include the orientation of
W21.sub.2, which is linked to alternate side-chain directions at
18.sub.1 and 38.sub.1, the burial of the larger tyrosine (Group 1)
versus histidine (Group 2) at 17.sub.1, and the packing of buried
His111.sub.2 (Group 1) against W21.sub.1. Other epitope residues
are indicated by numbered white circles. N38.sub.1 is glycosylated
in H3 and the H7 cluster. (e) Sequences of the 16 HA subtypes.
Circles indicate calculated binding energies: strong=blue,
intermediate=orange; weak=blue; unfavorable=black. Coloured
highlighting indicates sequence conservation within clusters and
groups. The network of inter-helical contacts that stabilize the
fusogenic structure.sup.29 are indicated below the sequences. FIG.
6E discloses "VDGW" as SEQ ID NO: 108, "IDGW" as SEQ ID NO: 106,
"INGW" as SEQ ID NO: 105, and "IAGW" as SEQ ID NO: 109.
[0031] FIG. 7 shows the prophylactic and therapeutic efficacy of
anti-H5 nAbs in mice. Mice were treated with different doses of nAb
either before or after lethal viral challenge. Prophylactic
efficacy (a, b, g and h). Mice were treated with anti-H5 nAbs or
control mAb 24 hour before lethal challenge by intranasally (i.n.)
with 10 median lethal doses (MLD.sub.50) of the H5N1 or H1N1s. (a)
Intra-peritoneal (i.p.) injection of 10 mg/kg of any of the three
nAbs provided complete protection of mice challenged with H5-VN04
(A/Vietnam/1203/04 (H5N1), Clade 1). A lower antibody dose (2.5
mg/kg) was also highly protective. (b) Prophylactic protection
against H5-HK97 (A/HongKong/483/97 (H5N1), Clade 0) virus was
observed in 80-100% of the mice treated with 10 mg/kg of any of the
three nAbs. (g) Any of the three nAbs (at 10 mg/kg of single
injection) provided complete protection of mice challenged with
H1-WSN33 (A/WSN/1933(H1N1)) viruses. (h) D8 and F10 completely
protected mice challenged with H1-PR34 (A/Puerto Rico/8/34 (H1N1))
when given at 10 mg/kg of single injection. A66 provided complete
protection of mice when 25 mg/kg of antibody was given as a single
injection. Therapeutic efficacy (c-f). Mice were inoculated with
H5-VN04 and injected with nAbs at 24, 48, 72 hpi (c, e and f) or
with H5-HK97 at 24 hpi (d). I.p. treatment with 15 mg/kg (a
therapeutically achievable dose in humans) of any of the 3 nAbs at
24 h post-inoculation (hpi) protected 80-100% of mice challenged
with 10-times the MLD.sub.50 of either H5-VN04 or H5-HK97
virus.
[0032] FIG. 8 illustrates SDS-PAGE and gel filtration analysis of
HA proteins. (a) Antibody 2A was obtained from a separate
HA1-targeted selection against the HA1 (residues 11-325) fragment
of H5-TH04 (left panel). H5 HA (H5-VN04 strain) used for library
selection is shown in the right panel, (b) H5-VN04 (H5) and scFv
F10 complex. HA0 was fully cleaved into HA1 and HA2 by
co-expression with furin (left panel). Complexes were formed by
first mixing H5 and F10 at a molar ratio of 1:10, and then purified
by gel filtration.
[0033] FIG. 9 show the binding of anti-H5 scFv-Fcs to H5 or HA1 by
ELISA and competition ELISA. (a) 1 ng/mL of anti-H5 scFv-Fcs
followed by HRP-anti-human IgG1 were used to detect the binding of
anti-H5 scFv-Fcs to HA1 (H5-TH04) or H5 (H5-VN04) coated on an
ELISA plate. An antibody selected against the HA1 subunit, mAb 2A
scFv-Fc, bound to both HA1 and H5. The 10 potent neutralizing
scFv-Fcs bound to H5 but not HA1. (b) 10.sup.12 pfu of anti-H5
phage-scFvs were mixed with 5 ng/mL of anti-H5 scFv-Fcs and added
to H5 (H5-VN04)-coated plates, washed, and followed by HRP-anti-M13
to detect phage-scFvs bound to H5. mAb 2A-Fc did not compete for
the epitope recognized by the 10 H5-selected Abs. All H5-selected
scFv-Fcs cross-competed. Of these, Ab F10 (phage-scFv) binding to
the H5 trimer was the least inhibited by the other scFv-Fcs
suggesting that it has the highest affinity among all Abs
tested.
[0034] FIG. 10 shows the kinetic and thermodynamic characterization
of the binding of H5 to nAbs D8, F10 and A66-IgG1s. nAbs were
captured on a CM4 chip via anti-human IgG1; trimeric H5 (H5-VN04)
at various concentrations (20, 10, 5, 2.5, 2.5, 1.25, 0.625 nM) was
injected over the chip surface. Binding kinetics were evaluated
using a 1:1 Langmuir binding model. The recorded binding curves
(with blank reference subtracted) and the calculated curves are
closely superimposable. Each ka, kd and K.sub.D value represents
the mean and standard error of three experiments.
[0035] FIG. 11 shows the phylogenetic relationships and sequence
comparison among HA subtypes. Phylogenetic tree of the 16 HA
subtypes of influenza A viruses based on amino-acid sequences. Four
clusters of HA subtypes are shaded in different colors. The
sequences used for analysis were: H1 (A/South Carolina/1/1918), H2
(A/Japan/305/1957), H3 (A/Aichi/2/1968), H4
(A/duck/Czechoslovakia/56), H5 (A/VietNam1203/2004), H6
(A/chicken/California/431/00), H7 (A/Netherland/219/03), H8
(A/turkey/Ontario/6118/68), H9 (A/swine/HK/9/98), H10
(A/chicken/Germany/N49), H11 (A/duck/England/56), H12
(A/duck/Alberta/60/76), H13 (A/gull/Maryland/704/77), H14
(A/mallard/Astrakhan/263/1982), H15 (A/shearwater/West
Australia/2576/79) and H16 (A/black-headed gull/Sweden/2/99).
[0036] FIG. 12 shows viral titers in lung, spleen and brain of mice
treated with anti-H5 nAbs after H5-VN04 challenge. BALB/c mice
(n=5) were treated by i.p. injection of 15 mg/kg of mAb at 24, 48
or 72 hrs after i.n. infection with 10 MLD.sub.50 of H5-VN04. Viral
titers were determined in lung, brain, and spleen collected at 96
hpi. Data are displayed in box-and-whiskers form in which the box
extends from the 25.sup.th to the 75.sup.th percentile, with a
horizontal line at the median. Whiskers above and below the box
indicate the extreme values. Results of Student T-test statistic
analysis are noted with a single star (*) for p<0.05, and double
stars (**) for p<0.01. The arrows crossing the Y axis indicate
the detection limit of the titration.
[0037] FIG. 13 shows FACS analysis of anti-H5 nAbs binding to all
H1 Cluster HAs including H1, H2, H5, H6, H11, H13 and H16. 293T
cells were transiently transfected with different HA-expressing
plasmids, and mAb binding to the HA-expressing cells was analyzed
by FACS. The anti-SARS mAb 80R was used as a control. 2A-Fc is an
H5 HA specific antibody. Lack of binding to a Group 2 HA, H7, was
also shown. Viral strain details of H11, H13 and H16 are: H11-MP74
(A/Duck/memphis/546/74 (H11N9)), H13-MD77 (A/Gull/MD/704/77
(H13N6)) and H16-DE06 (A/Shorebird/DE/172/06 (H16N3)).
DETAILED DESCRIPTION OF THE INVENTION
[0038] Influenza A is a negative-sense, single-stranded RNA virus,
with an eight-segment genome encoding 10 proteins. It belongs to
the family Orthomyxoviridae which includes the genera of influenza
virus A, B and C as defined by the antigenicity of the nucleocapsid
and matrix proteins. Generally, influenza A virus is associated
with more severe disease in humans. Influenza A virus is further
subtyped by two surface proteins, hemagglutinin (HA) which attaches
the virion to the host cell for cell entry, and neuraminidase (NA)
which facilitates the spread of the progeny virus by cleaving the
host sialic acid attached to the progeny virus or cell surface.
[0039] There are 16 HA subtypes and 9 NA subtypes which make up all
subtypes of influenza A viruses by various combinations of HA and
NA. All combinations of the 16 HA and 9 NA virus subtypes are found
in water fowl. Of the hundreds of strains of avian influenza A
viruses, only four are known to have caused human infections: H5N1,
H7N3, H7N7 and H9N2. In general, human infection with these viruses
has resulted in mild symptoms and very little severe illness: there
has been only one fatal case of pneumonia caused by H7N7. However,
the exception is the highly pathogenic H5N1 virus, for which there
is no natural immunity in humans. The infidelity of the RNA
polymerase and the selective pressure of host immunity can lead to
the accumulation of mutations and change in surface antigenicity of
these proteins. This antigenic change is called antigenic drift. In
addition, as a result of its segmented genome, shuffling of gene
segments can occur if two different subtypes of influenza A virus
infect the same cell. For example, if a human H3N2 virus and an
avian H5N1 virus co-infect a human or other member of a mammalian
species, such an event can produce a novel H5N2. This novel virus
can then be efficiently transmitted from human to human because all
of most of the gene segments come from the human virus. Such
genetic reassortment would lead to a major antigen change, a
so-called antigenic shift, which would mean that most of the global
population would not have any neutralizing antibodies against the
reassortant virus. Such a situation, coupled with the high
mortality of influenza H5N1 pneumonia, is one of the most feared
scenarios in the field of public health.
[0040] Influenza virus hemagglutinin (HA) is the most variable
antigen of influenza virus, and is responsible for virus entry into
cells. It is synthesized as a trimeric precursor polypeptide HA0
which is post-translationally cleaved to two polypeptides HA1 and
HA2 linked by a single disulphide bond. The HA1 chain of HA is
responsible for the attachment of virus to the cell surface. HA2
mediates the fusion of viral and cell membranes in endosomes,
allowing the release of the ribonucleoprotein complex into the
cytoplasm. In contrast to HA1, the HA2 molecule represents a
relatively conserved part of HA. A second immunogenic influenza
protein is neuraminidase (NA). This tetrameric glycoprotein is
responsible for releasing virions from surface sialic acid on
producer cells, and may also have a role in promoting access to
target cells in the airways. Although neutralizing antibodies
against NA are protective in animals and man, there is a paucity of
data on their mechanisms of action. A recent report on the crystal
structure of N1 neuraminidase demonstrated the presence of a cavity
adjacent to its active site that may be exploited to develop new
anti-influenza drugs, including antibodies. This finding is
particularly important in light of the reports of emergence of drug
resistance to oseltamivir (Tamiflu) and zanamivir (Relenza) for
H5N1 viruses.
[0041] Both the HA1 and HA2 chains of HA are immunogenic and
antibodies reactive with both chains have been demonstrated after
natural infection in humans. While antibodies specific to HA1 are
mostly neutralizing, different mechanism of virus neutralization by
HA1 specific Mabs in vitro have been described including blocking
the receptor site on HA1, intracellular inhibition of virus-cell
fusion, or simultaneous attachment inhibition and virus-cell fusion
inhibition, depending on antibody concentration. Although less well
studied, inhibition of cell fusion by anti-HA2 antibodies has been
reported.
[0042] More than two decades ago, the HA molecule of the H3 subtype
was characterized by sequencing the HA of antigenic drift variants
and escape mutants, and the antigenic epitopes were mapped on the
molecule's three-dimensional structure. Since then, the antigenic
sites on H1, H2 and H5 of an avian pathogenic virus were mapped on
the three-dimensional structures of H3. After the outbreak of H5N1
infection in humans in Hong Kong in 1997 and the isolation of H9N2
virus from human cases in 1999, the X-ray structures of both
proteins were solved. However, antigenic drift of the 1997 swine
isolate (A/Duck/Singapore/3/97) that was used to solve the
structure, and more recently isolated highly pathogenic strains, is
significant. Indeed, there are 28 minor changes and two potentially
major changes between the swine isolate (A/Duck/Singapore/3/97) and
the HPAI H5N1 strain (A/Vietnam1203/04).
[0043] Phylogenetic analyses of the H5 HA genes from the 2004-2005
outbreak have shown two different lineages of HA genes, termed
clades 1 and 2. HPAI H5N1 strain (A/Vietnam1203/04) is a member of
Glade 1. Viruses in each of these clades are distributed in
non-overlapping geographic regions of Asia. The H5N1 viruses from
Indochina are tightly clustered within Glade 1, whereas H5N1
isolated from several surrounding countries are distinct from Glade
1 isolates, and belong in a more divergent Glade 2. Clade 1 viruses
were isolated from humans and birds in Vietnam, Thailand and
Cambodia but only from birds in Laos and Malaysia. The Glade 2
viruses were found in viruses isolated exclusively from birds in
China, Indonesia, Japan, and South Korea. The most recent
epidemiologic studies analyzed 82 H5N1 viruses isolated from
poultry throughout Indonesia and Vietnam, as well as 11 human
isolates from southern Vietnam together with sequence data
available in public databases, to address questions relevant to
virus introduction, endemicity and evolution.sup.36. Phylogenetic
analysis showed that all viruses from Indonesia form a distinct
sublineage of H5N1 genotype Z viruses, suggesting that this
outbreak likely originated from a single introduction via spread
throughout the country during the past two years. Continued virus
activities in Indonesia were attributed to transmission via poultry
movement within the country, rather than through repeated
introductions by bird migration. Within Indonesia and Vietnam, H5N1
viruses have evolved over time into geographically distinct groups
within each country.
[0044] Recently, the structure of HA from A/Vietnam1203/4 was
solved. Comparison of its amino acid sequences with the HA genes
from HPAI 2004 and 2005 isolates from Glade 1 and 2 viruses
identified 13 positions of antigenic variation that are mainly
clustered around the receptor binding domain, while the rest are
within the vestigial esterase domain. Regions of antigenic
variation have been identified in H1 and H3 serotypes (FIG. 1A).
For H1, these sites are designated Sa, Sb, Ca and Cb while for H3,
sites are designated A, B, C and D. Escape mutants of H5 HAs can be
clustered into three epitopes; site 1: an exposed loop (HA1
140-145) that overlaps antigenic sites A of H3 and Ca2 of H.sup.2;
site 2: HA1 residues 156 and 157 that corresponds to antigenic site
B in H3 serotypes; and 3) HA1 129-133, which is restricted to the
Sa site in H1 HAs and H9 serotypes. In the recent studies by Smith,
detection of positive selection at the amino acid level indicated
that eight residues in the HA proteins were under positive
selection (FIG. 1B). These residues include five in antigenic sites
A and E (positions 83, 86, 138, 140 and 141); two involved in
receptor binding (positions 129 and 175); and positions 156 is a
site for potential N-linked glycosylation that is near the
receptor-binding site. The results further revealed that three
residues in HA (Val 86, Ser 129 and Thr 156) were more frequently
observed in human isolates than in chicken or duck isolates and
likely represented early adaptation of H5N1 genotype Z to humans.
Another important finding from these studies is that the
phylogenetic differences between the Indonesian and Vietnamese
sub-lineages was also reflected in significant differences in
antigenic cross-reactivity between these two group of viruses.
Specifically, viruses from Indonesia did not react to ferret
antisera against A/Vietnam1203/04, and representative viruses from
Vietnam did not react with ferret antisera against Indonesian
viruses IDN/5/06 and Dk/IDN/MS/04. These findings are in agreement
with earlier studies with immune human serum and human 1997 and
2003 H5N1 viruses that these strains were not only phylogenetically
but also antigenically distinct. Thus, natural variation as well as
escape mutants suggests that continued evolution of the virus
should impact the decision on which strain(s) should be used for
passive and active immunization.
[0045] The instant invention provides methods for the
identification, production and characterization of human
anti-influenza monoclonal antibodies
Identification and Characterization of scFvs and Monoclonal
Antibodies
[0046] High affinity, cross-subtype, broadly-neutralizing human
anti-HA mAbs have been identified. These nAbs inhibit the
post-attachment fusion process by recognizing a novel and highly
conserved neutralizing epitope within the stem region at a point
where key elements of the conformational change--the fusion peptide
and the exposed surface of helix .alpha.A--are brought into close
apposition. Structural and sequence analysis of all 16 HA subtypes
points to the existence of only two variants of this epitope,
corresponding to the two phylogenetic groupings of HA (Groups 1 and
2). These results raise the possibility that a small cocktail of
nAbs derived from a subset of each group could provide broad
protection against both seasonal and pandemic influenza.
[0047] A recent report utilized immune cells from H5N1-infected
patients to isolate anti-HA nAbs. However, their epitopes and modes
of action were not reported. Remarkably, we repeatedly isolated
nAbs that utilizes the same VH germline gene, IGHV1-69*01, and
encodes a CDR3 loop containing a tyrosine at an equivalent position
to Y102, from a non-immune library. This suggests that broad
anti-HA cross-immunity pre-exists in the H5-naive population,
possibly due to previous exposure to H1, and, for library donors
born before 1968, H2 subtypes. The recurrent use of this germline
VH segment, the commonality of the CDR3 tyrosine introduced through
N insertion and/or germline D gene assembly, and the promiscuous
use of VL genes by the nAbs discovered in both studies, suggest
that the precursor frequency of rearranged VH segments that could
recognize this epitope is significant. This raises the possibility
that with suitable exposure to the F10 epitope identified here,
these broad-spectrum nAbs may be readily induced in vivo. While the
genetic and structural complexity of nAbs required to provide
universal protection against virus subtypes in both groups is still
unknown, our data would seem to point to a surprisingly simple
solution.
[0048] Three unique anti-HA-1 scFvs were identified by sequencing
analysis of the 58 HA-1 positive clones. These scFvs were
designated as 38 B and 1C. The VH and VL amino acid sequence of 2A
is shown in FIG. 3.
[0049] Ten unique anti-HA0 scFvs were identified by sequencing
analysis of the 97 HA0 positive clones. These scFvs were designated
as 7, 8, 10, 17, 40, 66, 80, 88, 90, and 98. Six different VH and
10 different VL genes were revealed. Some scFvs shared the same VH
gene. Five out of the six different VH genes belonged to the
IGHV1-69 gene family. Three out of ten VL genes were kappa
chain.
[0050] 2A scFv is an moderate neutralizing antibody, 38B and 1C are
non-neutralizing antibodies. Ten scFvs, 7, 8, 10, 17, 40, 66, 80,
88, 90, and 98 are potent neutralizing antibodies. (FIG. 6)
[0051] The nucleic acid and amino acid sequence of the neutralizing
influenza antibodies are provided below:
TABLE-US-00001 TABLE 1A Antibody 2A Variable Region nucleic acid
sequences V.sub.H chain of 2A (SEQ ID NO: 1)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTC-
TGGAGG
CACCTTCAGTGACAATGCTATCAGCTGGGTGCGACAGGCCCCAGGACAAGGGCTTGAGTGGATGGGGGGCATCA-
TTCCTA
TCTTTGGAAAACCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACTGCGGACGAATCCACGAGCACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGATTCAGACGCGTATTACTA-
TGGTTC GGGGGGTATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA V.sub.L
chain of 2A (SEQ ID NO: 3)
CTGCCTGTGCTGACTCAATCATCCTCTGCCTCTGCTTCCCTGGGATCCTCGGTCAAGCTCACCTGCACTCTGAG-
CAGTGG
GCATAGTAACTACATCATCGCATGGCATCAACAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTTAATA-
GTGATG
GCAGCCACACCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGACCGCTACCTCACC-
ATCTCC
AACCTCCAGTCTGAGGATGAGGCTAGTTATTTCTGTGAGACCTGGGACACTAAGATTCATGTCTTCGGAACTGG-
GACCAA GGTCTCCGTCCTCAG
TABLE-US-00002 TABLE 1B Antibody 2A Variable Region amino acid
sequences V.sub.H chain of 2A (SEQ ID NO: 2)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNAISWVRQAPGQGLEWMGGIIPIFGKPNYAQKFQGRVTITADE-
STSTAY MDLRSLRSEDTAVYYCARDSDAYYYGSGGMDVWGQGTLVTVSS V.sub.L chain of
2A (SEQ ID NO: 4)
LPVLTQSSSASASLGSSVKLTCTLSSGHSNYIIAWHQQQPGKAPRYLMKVNSDGSHTKGDGIPDRFSGSSSGAD-
RYLTIS NLQSEDEASYFCETWDTKIHVFGTGTKVSVL
TABLE-US-00003 TABLE 1C Antibody D7 Variable Region nucleic acid
sequences V.sub.H chain of D7 (SEQ ID NO: 5)
CAGGIGCAGCTGGtGCAgTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCC-
TGGAGG
TATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGGGTCA-
TCCCTT
TGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAACACA-
GCCTAC
ATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGTGGTTACCATTTTAG-
GAGTCA CTTTGACTCCTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA V.sub.L chain of
D7 (SEQ ID NO: 9)
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGCGTCTCCGGGGAAGACGGTGACCATCTCCTGCACCGGCAG-
CAGTGG
CAACATTGCCGCCAACTATGTGCAGTGGTACCAACAACGTCCGGGCAGTGCCCCCACTACTGTGATCTATGAGG-
ATGACC
GAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGGTCCTCCAACTCTGCCTCCCTCACCATC-
TCAGGA
CTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGATACCAACAATCATGCTGTGTTCGGAGGAGG-
CACCCA CCTGACCGTCCTC
TABLE-US-00004 TABLE 1D Antibody H98 Variable Region nucleic acid
sequences V.sub.H chain of H98 (SEQ ID NO: 7)
CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCC-
TGGAGG
TATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGGGTCA-
TCCCTT
TGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAACACA-
GCCTAC
ATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGTGGTTACCATTTTAG-
GAGTCA CTTTGACTCCTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA V.sub.L chain of
H98 (SEQ ID NO: 11)
TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGAAACACGGGCAGAGGGTCACCATCTCTTGTTCTGGAGG-
CACCTC
CAACATCGGACGTAATCATGTTAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTA-
ATGAAC
AGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAATCTGGCACCTCCGCCTCCCTGGCCGTGAGTGGG-
CTCCAG
TCTGAGGATGAGGCTGATTATTACTGTGCATCATGGGATGACAACTTGAGTGGTTGGGTGTTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00005 TABLE 1E Antibody D7 and H98 Variable Region chain
amino acid sequences V.sub.H chain of D7 and H98 (SEQ ID NO: 6)
QVQLVQSGAEVKKPGSSVKVSCKAPGGIFNTNAFSWVRQAPGQGLEWVGGVIPLFRTASYAQNVQGRVTITADE-
STNTAY MELTSLRSADTAVYYCARSSGYHFRSHFDSWGLGTLVTVSS V.sub.L chain of
D7 (SEQ ID NO: 8)
NFMLTQPHSVSASPGKTVTISCTGSSGNIAANYVQWYQQRPGSAPTTVIYEDDRRPSGVPDRFSGSIDRSSNSA-
SLTISG LKTEDEADYYCQTYDTNNHAVFGGGTHLTVL V.sub.L chain of H98 (SEQ ID
NO: 10)
SYELTQPPSASGKHGQRVTISCSGGTSNIGRNHVNWYQQLPGTAPKLLIYSNEQRPSGVPDRFSGSKSGTSASL-
AVSGLQ SEDEADYYCASWDDNLSGWVFGGGTKLTVL
TABLE-US-00006 TABLE 1F Antibody D8 Variable Region nucleic acid
sequences V.sub.H chain of D8 (SEQ ID NO: 13)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTC-
TGGAGG
CACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGCATCA-
CCGGAA
TGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTCACGAGCACA-
GCCTAC
ATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTGTATTACTATGAGAG-
TAGTCT TGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG V.sub.L chain of
D8 (SEQ ID NO: 17)
CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAAC-
CAGCAG
TGACGTTGGTGGTTATAACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATG-
AGGTCA
CTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCT-
GGGCTC
CAGGCTGAGGATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCACAGTGCTTATGTCTTCGGAACTGGGAC-
CAAGGT CACCGTCCTG
TABLE-US-00007 TABLE 1G Antibody D80 Variable Region nucleic acid
sequences V.sub.H chain of D80 (SEQ ID NO: 15)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCA,GGCTTC-
TGGAGG
CACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGCATCA-
CCGGAA
TGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTCACGAGCACA-
GCCTAC
ATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTGTATTACTATGAGAG-
TAGTCT TGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG V.sub.K chain of
D80 (SEQ ID NO: 19)
GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC-
CAGTCA
GAGTCTTAGCAGCAAGTACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTG-
CATCCA
GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGTAGA-
CTGGAG
CCTGAAGATTTTGCAGTGTATTCCTGTCAGCAGTATGATGGCGTACCTCGGACGTTCGGCCAAGGGACCACGGT-
GGAAAT CAAA
TABLE-US-00008 TABLE 1H Antibody D8 and D80 Variable Region chain
amino acid sequences V.sub.H chain of D8 and D80 (SEQ ID NO: 12)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAFTWVRQAPGQGLEWMGGITGMFGTANYAQKFQGRVTITADE-
LTSTAY MELSSLTSEDTALYYCARGLYYYESSLDYWGQGTLVTVSS V.sub.L chain of D8
(SEQ ID NO: 14)
QSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNSVSWYQQHPGKAPKLMIYEVTKRPSGVPDRFSASKSGNTAS-
LTVSGL QAEDEADYFCCSYAGHSAYVFGTGTKVTVL V.sub.K chain of D80 (SEQ ID
NO: 16)
EIVLTQSPGTLSLSPGERATLSCRASQSLSSKYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL-
TISRLE PEDFAVYSCQQYDGVPRTFGQGTTVEIK
TABLE-US-00009 TABLE 1I Antibody F10 Variable Region nucleic acid
sequences V.sub.H chain of F10 (SEQ ID NO: 21)
CAGGTGCAGCTGGTGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTC-
TGAAGT
CACCTTCAGTAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGGATCA-
GCCCTA
TGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAGAGTCACCATTACCGCGGACCAGTCCACGAGGACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCTTCTTACATTTGTTC-
TGGTGG AACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA V.sub.L
chain of F10 (SEQ ID NO: 25)
CAGCCTGGGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCACACTCACCTGCACTGGGAA-
CAGCAA
CAATGTTGGCAACCAAGGAGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTCCTATCCTACAGGA-
ATAATG
ACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAACACAGCCTCCCTGACCATTACTGGA-
CTCCAG
CCTGAGGACGAGGCTGACTATTACTGCTCAACATGGGACAGCAGCCTCAGTGCTGTGGTATTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00010 TABLE 1J Antibody E90 Variable Region nucleic acid
sequences V.sub.H chain of E90 (SEQ ID NO: 23)
CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTC-
TGAAGT
CACCTTCAGTAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGGATCA-
GCCCTA
TGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAGAGTCACCATTACCGCGGACCAGTCCACGAGGACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCTTCTTACATTTGTTC-
TGGTGG AACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA V.sub.L
chain of E90 (SEQ ID NO: 27)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC-
AAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAT-
CCAGTT
TGCAAAGAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTAGCAGCCTG-
CAGCCT
GAAGATTTTGCAGTGTATTACTGTCAGCAGTATGATAGTTCACCGTACACTTTTGGCCAGGGGACCAAGGTAGA-
GATCAA A
TABLE-US-00011 TABLE 1K Antibody F10 and E90 Variable Region amino
acid sequences V.sub.H chain of F10 and E90 (SEQ ID NO: 18)
QVQLVQSGAEVKKPGSSVKVSCTSSEVTFSSFAISWVRQAPGQGLEWLGGISPMFGTPNYAQKFQGRVTITADQ-
STRTAY MDLRSLRSEDTAVYYCARSPSYICSGGTCVFDHWGQGTLVTVSS V.sub.L chain
of F10 (SEQ ID NO: 20)
QPGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNDRPSGISERFSASRSGNTASL-
TITGLQ PEDEADYYCSTWDSSLSAVVFGGGTKLTVL V.sub.L chain of E90 (SEQ ID
NO: 22)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQRGVPSRFSGSGSGTDFTLT-
ISSLQP EDFAVYYCQQYDSSPYTFGQGTKVEIK
TABLE-US-00012 TABLE 1L Antibody G17 Variable Region nucleic acid
sequences V.sub.H chain of G17 (SEQ ID NO: 29)
CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGACTTC-
TGGAGT
CACCTTCAGCAGCTATGCTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCA-
TCGGTG
TCTTTGGTGTACCAAAGTACGCGCAGAACTTCCAGGGCAGAGTCACAATTACCGCGGACAAACCGACGAGTACA-
GTCTAC
ATGGAGCTGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGCCCGGGTACTACGTAGG-
AAAGAA TGGTTTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.L chain
of G17 (SEQ ID NO: 31)
TCCTATGAGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCATACTCACCTGCACTGGAGA-
CAGCAA
CAATGTTGGCCACCAAGGTACAGCTTGGCTGCAACAACACCAGGGCCACCCTCCCAAACTCCTATCCTACAGGA-
ATGGCA
ACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAATACAGCCTCCCTGACCATTATTGGA-
CTCCAG
CCTGAGGACGAGGCTGACTACTACTGCTCAGTATGGGACAGCAGCCTCAGTGCCTGGGTGTTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00013 TABLE 1M Antibody G17 Variable Region amino acid
sequences V.sub.H chain of G17 (SEQ ID NO: 24)
QVQLVQSGAEVKKPGASVKVSCKTSGVTFSSYAISWVRQAPGQGLEWMGGIIGVFGVPKYAQNFQGRVTITADK-
PTSTVY MELNSLRAEDTAVYYCAREPGYYVGKNGFDVWGQGTMVTVSS V.sub.L chain of
G17 (SEQ ID NO: 26)
SYELTQPPSVSKGLRQTAILTCTGDSNNVGHQGTAWLQQHQGHPPKLLSYRNGNRPSGISERFSASRSGNTASL-
TIIGLQ PEDEADYYCSVWDSSLSAWVFGGGTKLTVL
TABLE-US-00014 TABLE 1N Antibody 1140 Variable Region nucleic acid
sequences V.sub.H chain of H40 (SEQ ID NO: 33)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTCAGTGAAGGTCTCATGTAAGGCTTC-
TGGATA
CACCTTCACCGGTTATTATATTCACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGTTGGATCA-
ACCCTA
TGACTGGTGGCACAAACTATGCACAGAAGTTTCAGGTCTGGGTCACCATGACCCGGGACACGTCCATCAACACA-
GCCTAC
ATGGAGGTGAGCAGGCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGGGGCTTCCGTATTACGATA-
TTTTGA CTGGCAGCCCGAGGCTCTTGATATCTGGGGCCTCGGGACCACGGTCACCGTCTCCTCA
V.sub.L chain of H40 (SEQ ID NO: 35)
CAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGCATTCCCTGTGGGGGGAA-
CAACAT
TGGAGGCTACAGTGTACACTGGTACCAACAAAAGCCGGGCCAGGCCCCCCTCTTGGTCATTTATGACGATAAAG-
ACCGGC
CCTCAGGGATCCCTGAGCGATTCTCTGGCGCCAACTCTGGGAGCACGGCCACCCTGACAATCAGCAGGGTCGAA-
GCCGGG
GATGAGGGCGACTACTACTGTCAGGTGTGGGATAGTGGTAATGATCGTCCGCTGTTCGGCGGAGGGACCAAGCT-
GACCGT CCTA
TABLE-US-00015 TABLE 1O Antibody 1140 Variable Region amino acid
sequences V.sub.H chain of H40 (SEQ ID NO: 28)
QVQLVQSGAEVRKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWTNPMTGGTNYAQKFQVWVTMTRDT-
SINTAY MEVSRLTSDDTAVYYCARGASVLRYFDWQPEALDIWGLGTTVTVSS V.sub.L chain
of H40 (SEQ ID NO: 30)
QPVLTQPPSVSVAPGQTASIPCGGNNIGGYSVHWYQQKPGQAPLLVIYDDKDRPSGIPERFSGANSGSTATLTI-
SRVEAG DEGDYYCQVWDSGNDRPLEGGGTKLTVL
TABLE-US-00016 TABLE 1P Antibody A66 Variable Region nucleic acid
sequences V.sub.H chain of A66 (SEQ ID NO: 37)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTC-
TGGAGG
CCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCA-
GCCCTA
TCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAACACA-
GCCAAC
ATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTTTCCTCCTACCAACC-
GAATAA TGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.K
chain of A66 (SEQ ID NO: 39)
GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC-
CAGTCA
GAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCAT-
CCAACA
GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTG-
GAGCCT
GAAGATTTTGCAGTCTATTTCTGTCAGCAGTATGGTAGCTCACCTCAATTCGGCCAAGGGACACGACTGGAGAT-
TAAA
TABLE-US-00017 TABLE 1Q Antibody A66 Variable Region amino acid
sequences V.sub.H chain of A66 (SEQ ID NO: 32)
QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGGISPIFRTPKYAQKFQGRVTITADE-
STNTAN MELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVSS V.sub.K chain of
A66 (SEQ ID NO: 34)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT-
ISRLEP EDFAVYFCQQYGSSPQFGQGTRLEIK
TABLE-US-00018 TABLE 1R Antibody E88 Variable Region nucleic acid
sequences V.sub.H chain of E88 (SEQ ID NO: 40)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTC-
TGGAGG
CCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCA-
GCCCTA
TCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAACACA-
GCCAAC
ATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTTTCCTCCTACCAACC-
GAATAA TGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.L
chain of E88 (SEQ ID NO: 42)
CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAG-
CAGCTC
CAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTA-
ATAATC
AGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCAGGCACCTCAGCCTCCCTGGCCATCATTGGA-
CTCCGG
CCTGAGGATGAAGCTGATTATTACTGTCAGTCGTATGACAGCAGGCTCAGTGCTTCTCTCTTCGGAACTGGGAC-
CACGGT CACCGTCCTC
TABLE-US-00019 TABLE 1S Antibody E88 Variable Region amino acid
sequences V.sub.H chain of E88 (SEQ ID NO: 36)
QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGGISPIFRTPKYAQKFQGRVTITADE-
STNTAN MELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVSS V.sub.L chain of
E88 (SEQ ID NO: 38)
LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSRSGTSASL-
AIIGLR PEDEADYYCQSYDSRLSASLFGTGTTVTVL
The amino acid sequences of the heavy and light chain
complementarity determining regions of the neutralizing influenza
antibodies are shown in Table 2 below and FIG. 3.
TABLE-US-00020 TABLE 2 Antibody CDR1 CDR2 CDR3 CONSENSUS SYAFS (SEQ
ID NO: 43) GIIPMFGTPNYAQKFQG SSGYYYG GGFDV (SEQ ID NO: 51) (SEQ ID
NO: 59) D7/H98VH TNAFS (SEQ ID NO: 44) GVIPLFRTASYAQNVQG
SSGYHFGRSHFDS (SEQ ID NO: 52) (SEQ ID NO: 60) D8/D80VH AYAFT (SEQ
ID NO: 45) GIIGMFGTANYAQKFQG GLYYYESSLDY (SEQ ID NO: 53) (SEQ ID
NO: 61) F10/90VH SFAIS (SEQ ID NO: 46) GISPMFGTPNYAQKFQG
SPSYICSGGTCVFDH (SEQ ID NO: 54) (SEQ ID NO: 62) G17VH SYAIS (SEQ ID
NO: 47) GIIGVFGVPKYAQKFQG EPGYYVGKNGFDV (SEQ ID NO: 55) (SEQ ID NO:
63) H40VH GYYIH (SEQ ID NO: 48) WINPMTGGTNYAQKFQV GASVLRYFDWQPEALDI
(SEQ ID NO: 56) (SEQ ID NO: 64) A66VH MTAFT (SEQ ID NO: 49)
GISPIFRTPKYAQKFQG TLSSYQPNNDAFAI (SEQ ID NO: 57) (SEQ ID NO: 65)
2AVH DNAIS (SEQ ID NO: 50) GIIPIFGKPNYAQKFQG DSDAYYYGSGGMDV (SEQ ID
NO: 58) (SEQ ID NO: 66) CONSENSUS TGSSSNIGNYVA SNSDRPS (SEQ ID NO:
79) QSYDSLSAYV (SEQ ID NO: 67) (SEQ ID NO: 91) D7VL TGSSSNIAANYVQ
EDDRRPS (SEQ ID NO: 80) QSYDTNNHAV (SEQ ID NO: 68) (SEQ ID NO: 92)
DD8VL TGTSSDVGGYNSVS EVTKRPS (SEQ ID NO: 81) CSYAGHSAYV (SEQ ID NO:
69) (SEQ ID NO: 93) F10VL TGNSNNVGNQGAA RNNDRPS (SEQ ID NO: 82)
STWDSSLSAVV (SEQ ID NO: 70) (SEQ ID NO: 94) G17VH TGDSNNVGHQGTA
RNGNRPS (SEQ ID NO: 83) SVWDSSLSAWV (SEQ ID NO: 71) (SEQ ID NO: 95)
H40VH GGNNIGGYSVH DDKDRPS (SEQ ID NO: 84) QVWDSGNDRPL (SEQ ID NO:
72) (SEQ ID NO: 96) A66VH RASQSVSSYLA DASNRAT (SEQ ID NO: 85)
QQYGSSPQV (SEQ ID NO: 73) (SEQ ID NO: 97) D80VL RASQSLSSKYLA
GASSRAT (SEQ ID NO: 86) QQYDGVPRT (SEQ ID NO: 74) (SEQ ID NO: 98)
E88VL TGSSSNIGNYVA SNNQRPS (SEQ ID NO: 87) QSYDSRLSASL (SEQ ID NO:
75) (SEQ ID NO: 99) E90VK SGSSSNIGSNTVN AASSLQR (SEQ ID NO: 88)
QQYDSSPYT (SEQ ID NO: 76) (SEQ ID NO: 100) H98VL RASQSISSYLN
SNEQRPS (SEQ ID NO: 89) ASWDDNLSGWV (SEQ ID NO: 77) (SEQ ID NO:
101) 2AVL TLSSGHSNYIIA VNSDGSHTKGD ETWDTKIHV (SEQ ID NO: 78) (SEQ
ID NO: 90) (SEQ ID NO: 102)
[0052] Those skilled in the art will recognize that additional
scFvs and monoclonal antibodies having different binding affinities
may also be therapeutically effective. For example, antibodies and
scFvs having binding affinities ranging from about 1 pM to about
200 mM may also be therapeutically effective.
Neutralization of H5N1 with Anti-Influenza Antibodies.
[0053] H-5 pseudotyped viruses were incubated with bivalent scFv
and full length antibodies and the antibody-virus mixture was
contacted with 293T cells. Infectivity was quantified by measuring
luciferase activity in the target cells. The D7, D8, F10, G17, H40,
A66, D80, E88, E90, and H98, antibodies have potent neutralization
activity against H5. D8, F10 and A66 antibodies also crossed
neutralized H1N1, potently neutralized strain H1-SC/1918 and
moderately neutralized strain H1-PR-34 (FIGS. 4A and 4B).
Characterization of the 8, 10 and 66 Epitope.
[0054] Primary epitope mapping of 8, 10 and 66 binding to the
influenza hemagglutinin (HA) protein showed that epitopes of these
three antibodies were similar and are located at positions 307 on
HA1 and at positions 52, 59, 65, and 93 on HA2. This epitope is
comprised of the hemagglutinin protein which is not shed by the
virus. This is unlike most other known ant-influenza antibodies
which bind the neuraminidase protein which is shed by the
virus.
Structural Characterization of the nAb Epitope
[0055] The epitope and mode of binding of one of the nAbs, F10, by
solving the crystal structure of its scFv fragment in complex with
HA (H5-VN04) at 3.2 .ANG. resolution, and by mutagenesis. (FIG. 6
and Table 4)
[0056] In the complex, each H5 trimer binds three molecules of F10,
at symmetry-related sites, burying .about.1500 A.sup.2 of protein
surface per antibody; the structure of H5 itself is not
significantly altered by F10 binding. HA is synthesized as a single
chain, HA0, that is activated by proteolytic cleavage into two
subunits, HA1 and HA2. Cleavage leads to the burial of the "fusion
peptide" (comprising the first .about.21 residues of HA2) into the
membrane-proximal stem. F10 binding occurs exclusively in this
region (FIG. 6), making intimate contacts with the fusion peptide,
elements of HA1 and HA2 (both of which are integral to the
structure of this region) that lock the peptide into place in the
neutral pH conformation, as well as the large helical hairpin of
HA2 that undergoes a massive conformation change at acidic pH in
order to propel the fusion peptide from its viral membrane-proximal
pocket to the distal surface of the virus, where it can trigger
fusion with the endosomal membrane.
[0057] The heavy chain of F10 plays the major role in H5 binding,
utilizing the tips of its three complementarity-determining regions
(CDRs). Each F10 molecule make contacts with both the HA1 and HA2
subunits within a single monomer of the HA trimer (FIG. 6). The
contact region comprises a pocket formed by part of the HA2 fusion
peptide, with elements of HA1 on one side and an exposed face of
helix .alpha.A of HA2 on the other (FIGS. 4 and 6). A triad of
antibody residues--F55 and M54 from CDR H2, and Y102 from CDR
H3--form major contact points. The phenyl ring of F55 lies across a
flat surface formed by a prominent loop of the fusion peptide loop
(HA2 residues DGW 19.sub.2-21.sub.2 (H3 numbering scheme;
subscripts 1 and 2 refer to HA1 and HA2 chains)) and the aromatic
side-chains of two flanking histidines (residues 18.sub.1 and
38.sub.1) and a tryptophan, W21.sub.2, which forms the back of the
pocket. The side-chain of M54 also contacts the aromatic rings of
W21.sub.2 and H38.sub.1, as well as the side-chain of I45.sub.2
from helix .alpha.A, while its main-chain carbonyl oxygen
hydrogen-bonds with the side-chain of H38.sub.1. Y102 inserts its
side-chain into a hydrophobic crevice created by four side-chains
of the .alpha.A helix, and also hydrogen-bonds to a backbone
carbonyl (D19.sub.2) of the fusion peptide. The CDR H1 loop makes
multiple contacts with the C-terminal end of helix .alpha.A and a
loop of HA1 at the base of the head region (FIGS. 6B and 6C).
[0058] In parallel, mutagenesis experiments were carried out on
helix .alpha.A to help define the epitope (FIG. 6). Mutations in 3
residues that directly contact the antibody: V52A/E, N53A and 156A,
significantly reduced or ablated antibody binding, while the
conservative mutation, V52L, had no effect. As controls, mutations
on a different exposed face of the helix, which does not contact
the antibody, had no effect on antibody binding (FIG. 6A-6D). Thus,
mutagenesis of the .alpha.A helix is fully consistent with the
epitope defined crystallographically for F10. Furthermore, the
other two nAbs, for which no structural data exist, showed an
almost identical mutant-nAb binding profile, indicative of a
closely overlapping epitope and consistent with competitive binding
(FIG. 6, FIG. 9). Taken together, we conclude that all three nAbs
neutralize virus by stabilizing the neutral pH conformation of HA
in a region that provides the trigger (most likely release of the
fusion peptide from its pocket) for conformational changes that
lead to the fusogenic state.
Antibodies
[0059] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically binds" or "immunoreacts with" is meant that the
antibody reacts with one or more antigenic determinants of the
desired antigen and does not react with other polypeptides.
Antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, dAb (domain antibody), single chain, F.sub.ab, F.sub.ab'
and F.sub.(ab')2 fragments, scFvs, and F.sub.ab expression
libraries.
[0060] A single chain Fv ("scFv") polypeptide molecule is a
covalently linked V.sub.H::V.sub.L heterodimer, which can be
expressed from a gene fusion including V.sub.H- and
V.sub.L-encoding genes linked by a peptide-encoding linker. (See
Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). A
number of methods have been described to discern chemical
structures for converting the naturally aggregated, but chemically
separated, light and heavy polypeptide chains from an antibody V
region into an scFv molecule, which will fold into a three
dimensional structure substantially similar to the structure of an
antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513;
5,132,405; and 4,946,778.
[0061] Very large naive human scFv libraries have been and can be
created to offer a large source of rearranged antibody genes
against a plethora of target molecules. Smaller libraries can be
constructed from individuals with infectious diseases in order to
isolate disease-specific antibodies. (See Barbas et al., Proc.
Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl.
Acad. Sci. USA 89:3175-79 (1992)).
[0062] In general, antibody molecules obtained from humans relate
to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from
one another by the nature of the heavy chain present in the
molecule. Certain classes have subclasses as well, such as
IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans, the light
chain may be a kappa chain or a lambda chain.
[0063] The term "antigen-binding site," or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." CDRs for the VH
and VL regions of the disclosed antibodies are shown in FIG. 3
respectively.
[0064] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin, an
scFv, or a T-cell receptor. Epitopic determinants usually consist
of chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. For example, antibodies may be raised against
N-terminal or C-terminal peptides of a polypeptide.
[0065] As used herein, the terms "immunological binding," and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
The strength, or affinity of immunological binding interactions can
be expressed in terms of the dissociation constant (K.sub.d) of the
interaction, wherein a smaller K.sub.d represents a greater
affinity Immunological binding properties of selected polypeptides
can be quantified using methods well known in the art. One such
method entails measuring the rates of antigen-binding site/antigen
complex formation and dissociation, wherein those rates depend on
the concentrations of the complex partners, the affinity of the
interaction, and geometric parameters that equally influence the
rate in both directions. Thus, both the "on rate constant"
(K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to a influenza epitope when
the equilibrium binding constant (K.sub.d) is .ltoreq.1 pM,
preferably 100 nM, more preferably .ltoreq.10 nM, and most
preferably .ltoreq.100 pM to about 1 pM, as measured by assays such
as radioligand binding assays or similar assays known to those
skilled in the art.
[0066] A influenza protein (e.g., HA or neuramindase) of the
invention, or a derivative, fragment, analog, homolog or ortholog
thereof, may be utilized as an immunogen in the generation of
antibodies that immunospecifically bind these protein
components.
[0067] Those skilled in the art will recognize that it is possible
to determine, without undue experimentation, if a human monoclonal
antibody has the same specificity as a human monoclonal antibody of
the invention by ascertaining whether the former prevents the
latter from binding to the HA protein of the influenza virus. If
the human monoclonal antibody being tested competes with the human
monoclonal antibody of the invention, as shown by a decrease in
binding by the human monoclonal antibody of the invention, then it
is likely that the two monoclonal antibodies bind to the same, or
to a closely related, epitope.
[0068] Another way to determine whether a human monoclonal antibody
has the specificity of a human monoclonal antibody of the invention
is to pre-incubate the human monoclonal antibody of the invention
with the influenza HA protein, with which it is normally reactive,
and then add the human monoclonal antibody being tested to
determine if the human monoclonal antibody being tested is
inhibited in its ability to bind the HA protein. If the human
monoclonal antibody being tested is inhibited then, in all
likelihood, it has the same, or functionally equivalent, epitopic
specificity as the monoclonal antibody of the invention. Screening
of human monoclonal antibodies of the invention, can be also
carried out by utilizing the influenza virus and determining
whether the test monoclonal antibody is able to neutralize the
influenza virus.
[0069] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs or orthologs thereof. (See, for example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference).
[0070] Antibodies can be purified by well-known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0071] The term "monoclonal antibody" or "MAb" or "monoclonal
antibody composition", as used herein, refers to a population of
antibody molecules that contain only one molecular species of
antibody molecule consisting of a unique light chain gene product
and a unique heavy chain gene product. In particular, the
complementarity determining regions (CDRs) of the monoclonal
antibody are identical in all the molecules of the population. MAbs
contain an antigen binding site capable of immunoreacting with a
particular epitope of the antigen characterized by a unique binding
affinity for it.
[0072] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes can be immunized in
vitro.
[0073] The immunizing agent will typically include the protein
antigen, a fragment thereof or a fusion protein thereof. Generally,
either peripheral blood lymphocytes are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103) Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells can be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0074] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies. (See Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63)).
[0075] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980). Moreover, in therapeutic applications of
monoclonal antibodies, it is important to identify antibodies
having a high degree of specificity and a high binding affinity for
the target antigen.
[0076] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods. (See Goding, Monoclonal Antibodies: Principles
and Practice, Academic Press, (1986) pp. 59-103). Suitable culture
media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma
cells can be grown in vivo as ascites in a mammal.
[0077] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0078] Monoclonal antibodies can also be made by recombinant DNA
methods, such as those described in U.S. Pat. No. 4,816,567. DNA
encoding the monoclonal antibodies of the invention can be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (see
U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
[0079] Fully human antibodies are antibody molecules in which the
entire sequence of both the light chain and the heavy chain,
including the CDRs, arise from human genes. Such antibodies are
termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by using trioma
technique; the human B-cell hybridoma technique (see Kozbor, et
al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to
produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96). Human monoclonal antibodies may be utilized and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc
Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells
with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96).
[0080] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries. (See
Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al.,
J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be
made by introducing human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al.,
Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368
856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et
al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature
Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev.
Immunol. 13 65-93 (1995).
[0081] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells which secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv (scFv) molecules.
[0082] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method, which includes deleting the J
segment genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0083] One method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. This
method includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain. In a further improvement on this procedure, a
method for identifying a clinically relevant epitope on an
immunogen, and a correlative method for selecting an antibody that
binds immunospecifically to the relevant epitope with high
affinity, are disclosed in PCT publication WO 99/53049.
[0084] The antibody can be expressed by a vector containing a DNA
segment encoding the single chain antibody described above.
[0085] These can include vectors, liposomes, naked DNA,
adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include
chemical conjugates such as described in WO 93/64701, which has
targeting moiety (e.g. a ligand to a cellular surface receptor),
and a nucleic acid binding moiety (e.g. polylysine), viral vector
(e.g. a DNA or RNA viral vector), fusion proteins such as described
in PCT/US 95/02140 (WO 95/22618) which is a fusion protein
containing a target moiety (e.g. an antibody specific for a target
cell) and a nucleic acid binding moiety (e.g. a protamine),
plasmids, phage, etc. The vectors can be chromosomal,
non-chromosomal or synthetic.
[0086] Preferred vectors include viral vectors, fusion proteins and
chemical conjugates. Retroviral vectors include moloney murine
leukemia viruses. DNA viral vectors are preferred. These vectors
include pox vectors such as orthopox or avipox vectors, herpesvirus
vectors such as a herpes simplex I virus (HSV) vector (see Geller,
A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA
Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press,
Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.
Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl.
Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (see LeGal
LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.
Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and
Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat.
Genet. 8:148 (1994).
[0087] Pox viral vectors introduce the gene into the cells
cytoplasm. Avipox virus vectors result in only a short term
expression of the nucleic acid. Adenovirus vectors,
adeno-associated virus vectors and herpes simplex virus (HSV)
vectors are preferred for introducing the nucleic acid into neural
cells. The adenovirus vector results in a shorter term expression
(about 2 months) than adeno-associated virus (about 4 months),
which in turn is shorter than HSV vectors. The particular vector
chosen will depend upon the target cell and the condition being
treated. The introduction can be by standard techniques, e.g.
infection, transfection, transduction or transformation. Examples
of modes of gene transfer include e.g., naked DNA, CaPO.sub.4
precipitation, DEAE dextran, electroporation, protoplast fusion,
lipofection, cell microinjection, and viral vectors.
[0088] The vector can be employed to target essentially any desired
target cell. For example, stereotaxic injection can be used to
direct the vectors (e.g. adenovirus, HSV) to a desired location.
Additionally, the particles can be delivered by
intracerebroventricular (icy) infusion using a minipump infusion
system, such as a SynchroMed Infusion System. A method based on
bulk flow, termed convection, has also proven effective at
delivering large molecules to extended areas of the brain and may
be useful in delivering the vector to the target cell. (See Bobo et
al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et
al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be
used include catheters, intravenous, parenteral, intraperitoneal
and subcutaneous injection, and oral or other known routes of
administration.
[0089] These vectors can be used to express large quantities of
antibodies that can be used in a variety of ways. For example, to
detect the presence of an influenza virus in a sample. The antibody
can also be used to try to bind to and disrupt influenza virus cell
membrane fusion.
[0090] Techniques can be adapted for the production of single-chain
antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted
for the construction of F.sub.ab expression libraries (see e.g.,
Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and
effective identification of monoclonal F.sub.ab fragments with the
desired specificity for a protein or derivatives, fragments,
analogs or homologs thereof. Antibody fragments that contain the
idiotypes to a protein antigen may be produced by techniques known
in the art including, but not limited to: (i) an F.sub.(ab'2)
fragment produced by pepsin digestion of an antibody molecule; (ii)
an F.sub.ab fragment generated by reducing the disulfide bridges of
an F.sub.(ab'2) fragment; (iii) an F.sub.ab fragment generated by
the treatment of the antibody molecule with papain and a reducing
agent and (iv) F.sub.v fragments.
[0091] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (see
U.S. Pat. No. 4,676,980), and for treatment of HIV infection (see
WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0092] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating influenza. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
(See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes,
J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can
be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. (See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989)).
[0093] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate).
[0094] Enzymatically active toxins and fragments thereof that can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are available for the
production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y and .sup.186Re.
[0095] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. (See WO94/11026).
[0096] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the resultant
antibodies or to other molecules of the invention. (See, for
example, "Conjugate Vaccines", Contributions to Microbiology and
Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press,
New York, (1989), the entire contents of which are incorporated
herein by reference).
[0097] Coupling may be accomplished by any chemical reaction that
will bind the two molecules so long as the antibody and the other
moiety retain their respective activities. This linkage can include
many chemical mechanisms, for instance covalent binding, affinity
binding, intercalation, coordinate binding and complexation. The
preferred binding is, however, covalent binding. Covalent binding
can be achieved either by direct condensation of existing side
chains or by the incorporation of external bridging molecules. Many
bivalent or polyvalent linking agents are useful in coupling
protein molecules, such as the antibodies of the present invention,
to other molecules. For example, representative coupling agents can
include organic compounds such as thioesters, carbodiimides,
succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes
and hexamethylene diamines This listing is not intended to be
exhaustive of the various classes of coupling agents known in the
art but, rather, is exemplary of the more common coupling agents.
(See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984);
Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta
et al., Science 238:1098 (1987)). Preferred linkers are described
in the literature. (See, for example, Ramakrishnan, S. et al.,
Cancer Res. 44:201-208 (1984) describing use of MBS
(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S.
Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide
derivative coupled to an antibody by way of an oligopeptide linker.
Particularly preferred linkers include: (i) EDC
(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii)
SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.
#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce
Chem. Co., Cat. #24510) conjugated to EDC.
[0098] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NHS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, the linker SMPT contains a sterically
hindered disulfide bond, and can form conjugates with increased
stability. Disulfide linkages, are in general, less stable than
other linkages because the disulfide linkage is cleaved in vitro,
resulting in less conjugate available. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
[0099] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0100] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction.
Use of Antibodies Against Influenza Virus
[0101] Methods for the screening of antibodies that possess the
desired specificity include, but are not limited to, enzyme linked
immunosorbent assay (ELISA) and other immunologically mediated
techniques known within the art.
[0102] Antibodies directed against a influenza virus protein such
as HA (or a fragment thereof) may be used in methods known within
the art relating to the localization and/or quantitation of a
influenza virus protein (e.g., for use in measuring levels of the
influenza virus protein within appropriate physiological samples,
for use in diagnostic methods, for use in imaging the protein, and
the like). In a given embodiment, antibodies specific to an
influenza virus protein, or derivative, fragment, analog or homolog
thereof, that contain the antibody derived antigen binding domain,
are utilized as pharmacologically active compounds (referred to
hereinafter as "Therapeutics").
[0103] An antibody specific for an influenza virus protein of the
invention can be used to isolate an influenza virus polypeptide by
standard techniques, such as immunoaffinity, chromatography or
immunoprecipitation. Antibodies directed against an influenza virus
protein (or a fragment thereof) can be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
[0104] Antibodies of the invention, including polyclonal,
monoclonal, humanized and fully human antibodies, may used as
therapeutic agents. Such agents will generally be employed to treat
or prevent an influenza virus-related disease or pathology (e.g.,
bird flu) in a subject. An antibody preparation, preferably one
having high specificity and high affinity for its target antigen,
is administered to the subject and will generally have an effect
due to its binding with the target. Administration of the antibody
may abrogate or inhibit or interfere with the internalization of
the virus into a cell In this case, the antibody binds to the
target and masks a binding site of the naturally occurring ligand,
thereby blocking fusion the virus to the cell membrane inhibiting
internalization of the virus.
[0105] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target. The
amount required to be administered will furthermore depend on the
binding affinity of the antibody for its specific antigen, and will
also depend on the rate at which an administered antibody is
depleted from the free volume other subject to which it is
administered. Common ranges for therapeutically effective dosing of
an antibody or antibody fragment of the invention may be, by way of
nonlimiting example, from about 0.1 mg/kg body weight to about 50
mg/kg body weight. Common dosing frequencies may range, for
example, from twice daily to once a week.
[0106] Antibodies specifically binding an influenza virus protein
or a fragment thereof of the invention, as well as other molecules
identified by the screening assays disclosed herein, can be
administered for the treatment of an influenza virus-related
disorders in the form of pharmaceutical compositions. Principles
and considerations involved in preparing such compositions, as well
as guidance in the choice of components are provided, for example,
in Remington: The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.,
1995; Drug Absorption Enhancement: Concepts, Possibilities,
Limitations, And Trends, Harwood Academic Publishers, Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[0107] Where antibody fragments are used, the smallest inhibitory
fragment that specifically binds to the binding domain of the
target protein is preferred. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. (See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation
can also contain more than one active compound as necessary for the
particular indication being treated, preferably those with
complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition can comprise an
agent that enhances its function, such as, for example, a cytotoxic
agent, cytokine, chemotherapeutic agent, or growth-inhibitory
agent. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
[0108] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0109] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0110] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0111] An antibody according to the invention can be used as an
agent for detecting the presence of an influenza virus (or a
protein or a protein fragment thereof) in a sample. Preferably, the
antibody contains a detectable label. Antibodies can be polyclonal,
or more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., F.sub.ab, scFv, or F.sub.(ab)2) can be used. The
term "labeled", with regard to the probe or antibody, is intended
to encompass direct labeling of the probe or antibody by coupling
(i.e., physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently-labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of an analyte genomic DNA include
Southern hybridizations. Procedures for conducting immunoassays are
described, for example in "ELISA: Theory and Practice: Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press,
Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and
"Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-analyte protein antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
Pharmaceutical Compositions
[0112] The antibodies or agents of the invention (also referred to
herein as "active compounds"), and derivatives, fragments, analogs
and homologs thereof, can be incorporated into pharmaceutical
compositions suitable for administration. Such compositions
typically comprise the antibody or agent and a pharmaceutically
acceptable carrier. As used herein, the term "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Suitable carriers are described
in the most recent edition of Remington's Pharmaceutical Sciences,
a standard reference text in the field, which is incorporated
herein by reference. Preferred examples of such carriers or
diluents include, but are not limited to, water, saline, ringer's
solutions, dextrose solution, and 5% human serum albumin Liposomes
and non-aqueous vehicles such as fixed oils may also be used. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the compositions is contemplated. Supplementary active compounds
can also be incorporated into the compositions.
[0113] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0114] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0115] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0116] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0117] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0118] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0119] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0120] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0121] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0122] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Screening Methods
[0123] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) that modulate or otherwise interfere with
the fusion of an influenza virus to the cell membrane. Also
provided are methods of identifying compounds useful to treat
influenza infection. The invention also encompasses compounds
identified using the screening assays described herein.
[0124] For example, the invention provides assays for screening
candidate or test compounds which modulate the interaction between
the influenza virus and the cell membrane. The test compounds of
the invention can be obtained using any of the numerous approaches
in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds.
(See, e.g., Lam, 1997. Anticancer Drug Design 12: 145).
[0125] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and
most preferably less than about 4 kD. Small molecules can be, e.g.,
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art and can be
screened with any of the assays of the invention.
[0126] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt, et al., 1993.
Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc.
Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J.
Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell,
et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al.,
1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,
1994. J. Med. Chem. 37: 1233.
[0127] Libraries of compounds may be presented in solution (see
e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (see
Lam, 1991. Nature 354: 82-84), on chips (see Fodor, 1993. Nature
364: 555-556), bacteria (see U.S. Pat. No. 5,223,409), spores (see
U.S. Pat. No. 5,233,409), plasmids (see Cull, et al., 1992. Proc.
Natl. Acad. Sci. USA 89: 1865-1869) or on phage (see Scott and
Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249:
404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:
6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; and U.S. Pat.
No. 5,233,409.).
[0128] In one embodiment, a candidate compound is introduced to an
antibody-antigen complex and determining whether the candidate
compound disrupts the antibody-antigen complex, wherein a
disruption of this complex indicates that the candidate compound
modulates the interaction between an influenza virus and the cell
membrane. For example, the antibody may be monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, and H98 and the antigen may
be located on the HA protein of an influenza virus.
[0129] In another embodiment, at least one HA protein is provided,
which is exposed to at least one neutralizing monoclonal antibody.
Formation of an antibody-antigen complex is detected, and one or
more candidate compounds are introduced to the complex. If the
antibody-antigen complex is disrupted following introduction of the
one or more candidate compounds, the candidate compounds is useful
to treat a an influenza virus-related disease or disorder, e.g.
bird flu. For example, the at least one influenza virus protein may
be provided as an influenza virus molecule.
[0130] Determining the ability of the test compound to interfere
with or disrupt the antibody-antigen complex can be accomplished,
for example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to the
antigen or biologically-active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be
enzymatically-labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0131] In one embodiment, the assay comprises contacting an
antibody-antigen complex with a test compound, and determining the
ability of the test compound to interact with the antigen or
otherwise disrupt the existing antibody-antigen complex. In this
embodiment, determining the ability of the test compound to
interact with the antigen and/or disrupt the antibody-antigen
complex comprises determining the ability of the test compound to
preferentially bind to the antigen or a biologically-active portion
thereof, as compared to the antibody.
[0132] In another embodiment, the assay comprises contacting an
antibody-antigen complex with a test compound and determining the
ability of the test compound to modulate the antibody-antigen
complex. Determining the ability of the test compound to modulate
the antibody-antigen complex can be accomplished, for example, by
determining the ability of the antigen to bind to or interact with
the antibody, in the presence of the test compound.
[0133] Those skilled in the art will recognize that, in any of the
screening methods disclosed herein, the antibody may be a an
influenza virus neutralizing antibody, such as monoclonal antibody
D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98. Additionally,
the antigen may be a HA protein, or a portion thereof. In any of
the assays described herein, the ability of a candidate compound to
interfere with the binding between the D7, D8, F10, G17, H40, A66,
D80, E88, E90, and H98 monoclonal antibody and the HA protein
indicates that the candidate compound will be able to interfere
with or modulate the fusion of the influenza virus and the cell
membrane Moreover, because the binding of the HA protein to cell is
responsible for influenza virus entry into cells such candidate
compounds will also be useful in the treatment of a influenza virus
related disease or disorder, e.g. bird flu.
[0134] The screening methods disclosed herein may be performed as a
cell-based assay or as a cell-free assay. The cell-free assays of
the invention are amenable to use of both the soluble form or the
membrane-bound form of the HA proteins and fragments thereof. In
the case of cell-free assays comprising the membrane-bound forms of
the HA proteins, it may be desirable to utilize a solubilizing
agent such that the membrane-bound form of the proteins are
maintained in solution. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,
3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane
sulfonate (CHAPSO).
[0135] In more than one embodiment, it may be desirable to
immobilize either the antibody or the antigen to facilitate
separation of complexed from uncomplexed forms of one or both
following introduction of the candidate compound, as well as to
accommodate automation of the assay. Observation of the
antibody-antigen complex in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided that adds a domain that allows one or both
of the proteins to be bound to a matrix. For example, GST-antibody
fusion proteins or GST-antigen fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, that are then combined
with the test compound, and the mixture is incubated under
conditions conducive to complex formation (e.g., at physiological
conditions for salt and pH). Following incubation, the beads or
microtiter plate wells are washed to remove any unbound components,
the matrix immobilized in the case of beads, complex determined
either directly or indirectly. Alternatively, the complexes can be
dissociated from the matrix, and the level of antibody-antigen
complex formation can be determined using standard techniques.
[0136] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the antibody or the antigen (e.g. the can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated
antibody or antigen molecules can be prepared from
biotin-NHS(N-hydroxy-succinimide) using techniques well-known
within the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, other antibodies reactive with the antibody or
antigen of interest, but which do not interfere with the formation
of the antibody-antigen complex of interest, can be derivatized to
the wells of the plate, and unbound antibody or antigen trapped in
the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using such other antibodies reactive with the antibody or
antigen.
[0137] The invention further pertains to novel agents identified by
any of the aforementioned screening assays and uses thereof for
treatments as described herein.
Diagnostic Assays
[0138] Antibodies of the present invention can be detected by
appropriate assays, e.g., conventional types of immunoassays. For
example, a an assay can be performed in which a influenza protein
(e.g., HA1, HA 2 or neurominidase) or fragment thereof is affixed
to a solid phase. Incubation is maintained for a sufficient period
of time to allow the antibody in the sample to bind to the
immobilized polypeptide on the solid phase. After this first
incubation, the solid phase is separated from the sample. The solid
phase is washed to remove unbound materials and interfering
substances such as non-specific proteins which may also be present
in the sample. The solid phase containing the antibody of interest
bound to the immobilized polypeptide is subsequently incubated with
a second, labeled antibody or antibody bound to a coupling agent
such as biotin or avidin. This second antibody may be another
anti-influenza antibody or another antibody. Labels for antibodies
are well-known in the art and include radionuclides, enzymes (e.g.
maleate dehydrogenase, horseradish peroxidase, glucose oxidase,
catalase), fluors (fluorescein isothiocyanate, rhodamine,
phycocyanin, fluorescarmine), biotin, and the like. The labeled
antibodies are incubated with the solid and the label bound to the
solid phase is measured. These and other immunoassays can be easily
performed by those of ordinary skill in the art.
[0139] An exemplary method for detecting the presence or absence of
a influenza virus (in a biological sample involves obtaining a
biological sample from a test subject and contacting the biological
sample with a labeled monoclonal or scFv antibody according to the
invention such that the presence of the influenza virus is detected
in the biological sample.
[0140] As used herein, the term "labeled", with regard to the probe
or antibody, is intended to encompass direct labeling of the probe
or antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently-labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently-labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect an influenza virus in
a biological sample in vitro as well as in vivo. For example, in
vitro techniques for detection of an influenza virus include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. Furthermore, in vivo
techniques for detection of an influenza virus include introducing
into a subject a labeled anti-influenza virus antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0141] In one embodiment, the biological sample contains protein
molecules from the test subject. One preferred biological sample is
a peripheral blood leukocyte sample isolated by conventional means
from a subject.
[0142] The invention also encompasses kits for detecting the
presence of an influenza virus in a biological sample. For example,
the kit can comprise: a labeled compound or agent capable of
detecting an influenza virus (e.g., an anti-influenza scFv or
monoclonal antibody) in a biological sample; means for determining
the amount of an influenza virus in the sample; and means for
comparing the amount of an influenza virus in the sample with a
standard. The compound or agent can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect an influenza virus in a sample.
Passive Immunization
[0143] Passive immunization has proven to be an effective and safe
strategy for the prevention and treatment of viral diseases. (See
Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall,
Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10
(1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of
which are incorporated herein by reference)). Passive immunization
using neutralizing human monoclonal antibodies could provide an
immediate treatment strategy for emergency prophylaxis and
treatment of influenza such as bird flu while the alternative and
more time-consuming development of vaccines and new drugs in
underway.
[0144] Subunit vaccines potentially offer significant advantages
over conventional immunogens. They avoid the safety hazards
inherent in production, distribution, and delivery of conventional
killed or attenuated whole-pathogen vaccines. Furthermore, they can
be rationally designed to include only confirmed protective
epitopes, thereby avoiding suppressive T epitopes (see Steward et
al., J. Virol. 69:7668 (1995)) or immunodominant B epitopes that
subvert the immune system by inducing futile, non-protective
responses (e.g. "decoy" epitopes). (See Garrity et al., J. Immunol.
159:279 (1997)).
[0145] Moreover, those skilled in the art will recognize that good
correlation exists between the antibody neutralizing activity in
vitro and the protection in vivo for many different viruses,
challenge routes, and animal models. (See Burton, Natl. Rev.
Immunol. 2:706-13 (2002); Parren et al., Adv. Immunol. 77:195-262
(2001)). The data presented herein demonstrate that the D7, D8,
F10, G17, H40, A66, D80, E88, E90, and H98 human monoclonal
antibodies can be further developed and tested in in vivo animal
studies to determine its clinical utility as a potent viral entry
inhibitor for emergency prophylaxis and treatment of influenza.
Antigen-Ig Chimeras in Vaccination
[0146] It has been over a decade since the first antibodies were
used as scaffolds for the efficient presentation of antigenic
determinants to the immune systems. (See Zanetti, Nature 355:476-77
(1992); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35
(1995)). When a peptide is included as an integral part of an IgG
molecule (e.g., the 11A or 256 IgG1 monoclonal antibody described
herein), the antigenicity and immunogenicity of the peptide
epitopes are greatly enhanced as compared to the free peptide. Such
enhancement is possibly due to the antigen-IgG chimeras longer
half-life, better presentation and constrained conformation, which
mimic their native structures.
[0147] Moreover, an added advantage of using an antigen-Ig chimera
is that either the variable or the Fc region of the antigen-Ig
chimera can be used for targeting professional antigen-presenting
cells (APCs). To date, recombinant Igs have been generated in which
the complementarity-determining regions (CDRs) of the heavy chain
variable gene (V.sub.H) are replaced with various antigenic
peptides recognized by B or T cells. Such antigen-Ig chimeras have
been used to induce both humoral and cellular immune responses.
(See Bona et al., Immunol. Today 19:126-33 (1998)).
[0148] Chimeras with specific epitopes engrafted into the CDR3 loop
have been used to induce humoral responses to either HIV-1 gp120
V3-loop or the first extracellular domain (D1) of human CD4
receptor. (See Lanza et al., Proc. Natl. Acad. Sci. USA 90:11683-87
(1993); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35
(1995)). The immune sera were able to prevent infection of CD4
SupT1 cells by HIV-1MN (anti-gp120 V3C) or inhibit syncytia
formation (anti-CD4-D1). The CDR2 and CDR3 can be replaced with
peptide epitopes simultaneously, and the length of peptide inserted
can be up to 19 amino acids long.
[0149] Alternatively, one group has developed a "troybody" strategy
in which peptide antigens are presented in the loops of the Ig
constant (C) region and the variable region of the chimera can be
used to target IgD on the surface of B-cells or MHC class II
molecules on professional APCs including B-cells, dendritic cells
(DC) and macrophages. (See Lunde et al., Biochem. Soc. Trans.
30:500-6 (2002)).
[0150] An antigen-Ig chimera can also be made by directly fusing
the antigen with the Fc portion of an IgG molecule. You et al.,
Cancer Res. 61:3704-11 (2001) were able to obtain all arms of
specific immune response, including very high levels of antibodies
to hepatitis B virus core antigen using this method.
DNA Vaccination
[0151] DNA vaccines are stable, can provide the antigen an
opportunity to be naturally processed, and can induce a
longer-lasting response. Although a very attractive immunization
strategy, DNA vaccines often have very limited potency to induce
immune responses. Poor uptake of injected DNA by professional APCs,
such as dendritic cells (DCs), may be the main cause of such
limitation. Combined with the antigen-Ig chimera vaccines, a
promising new DNA vaccine strategy based on the enhancement of APC
antigen presentation has been reported (see Casares, et al., Viral
Immunol. 10:129-36 (1997); Gerloni et al., Nat. Biotech. 15:876-81
(1997); Gerloni et al., DNA Cell Biol. 16:611-25 (1997); You et
al., Cancer Res. 61:3704-11 (2001)), which takes advantage of the
presence of Fc receptors (Fc.gamma.Rs) on the surface of DCs.
[0152] It is possible to generate a DNA vaccine encoding an antigen
(Ag)-Ig chimera. Upon immunization, Ag-Ig fusion proteins will be
expressed and secreted by the cells taking up the DNA molecules.
The secreted Ag-Ig fusion proteins, while inducing B-cell
responses, can be captured and internalized by interaction of the
Fc fragment with Fc.gamma.Rs on DC surface, which will promote
efficient antigen presentation and greatly enhance antigen-specific
immune responses. Applying the same principle, DNA encoding
antigen-Ig chimeras carrying a functional anti-MHC II specific scFv
region gene can also target the immunogens to all three types of
APCs. The immune responses could be further boosted with use of the
same protein antigens generated in vitro (i.e., "prime and boost"),
if necessary. Using this strategy, specific cellular and humoral
immune responses against infection of influenza virus were
accomplished through intramuscular (i.m.) injection of a DNA
vaccine. (See Casares et al., Viral. Immunol. 10:129-36
(1997)).
Vaccine Compositions
[0153] Therapeutic or prophylactic compositions are provided
herein, which generally comprise mixtures of one or more monoclonal
antibodies or ScFvs and combinations thereof.
[0154] The prophylactic vaccines can be used to prevent an
influenza virus infection and the therapeutic vaccines can be used
to treat individuals following an influenza virus infection.
Prophylactic uses include the provision of increased antibody titer
to an influenza virus in a vaccination subject. In this manner,
subjects at high risk of contracting influenza can be provided with
passive immunity to an influenza virus
[0155] These vaccine compositions can be administered in
conjunction with ancillary immunoregulatory agents. For example,
cytokines, lymphokines, and chemokines, including, but not limited
to, IL-2, modified IL-2 (Cys125.fwdarw.Ser125), GM-CSF, IL-12,
.gamma.-interferon, IP-10, MIP1.beta., and RANTES.
Methods of Immunization
[0156] The vaccines of the present invention have superior
immunoprotective and immunotherapeutic properties over other
anti-viral vaccines
[0157] The invention provides a method of immunization, e.g.,
inducing an immune response, of a subject. A subject is immunized
by administration to the subject a composition containing a
membrane fusion protein of a pathogenic enveloped virus. The fusion
protein is coated or embedded in a biologically compatible
matrix.
[0158] The fusion protein is glycosylated, e.g. contains a
carbohydrate moiety. The carbohydrate moiety may be in the form of
a monosaccharide, disaccharide(s). oligosaccharide(s),
polysaccharide(s), or their derivatives (e.g. sulfo- or
phospho-substituted). The carbohydrate is linear or branched. The
carbohydrate moiety is N-linked or O-linked to a polypeptide.
N-linked glycosylation is to the amide nitrogen of asparagine side
chains and O-linked glycosylation is to the hydroxy oxygen of
serine and threonine side chains.
[0159] The carbohydrate moiety is endogenous to the subject being
vaccinated. Alternatively, the carbohydrate moiety is exogenous to
the subject being vaccinated. The carbohydrate moiety are
carbohydrate moieties that are not typically expressed on
polypeptides of the subject being vaccinated. For example, the
carbohydrate moieties are plant-specific carbohydrates. Plant
specific carbohydrate moieties include for example N-linked glycan
having a core bound .alpha.1,3 fucose or a core bound .beta.1,2
xylose. Alternatively, the carbohydrate moiety are carbohydrate
moieties that are expressed on polypeptides or lipids of the
subject being vaccinate. For example many host cells have been
genetically engineered to produce human proteins with human-like
sugar attachments.
[0160] For example, the fusion protein is a trimeric hemagglutinin
protein. Optionally, the hemagglutinin protein is produced in a
non-mammalian cell such as a plant cell. The subject is at risk of
developing or suffering from a viral infection. Enveloped viruses
include for example, epstein-barr virus, herpes simplex virus, type
1 and 2, human cytomegalovirus, human herpesvirus, type 8,
varicella zoster virus, hepatitis B virus, hepatitis C virus, human
immunodeficiency virus, influenza virus, measles virus, mumps
virus, parainfluenza virus, respiratory syncytial virus, rabies
virus, and rubella virus The methods described herein lead to a
reduction in the severity or the alleviation of one or more
symptoms of a viral infection. Infections are diagnosed and or
monitored, typically by a physician using standard methodologies A
subject requiring immunization is identified by methods know in the
art. For example subjects are immunized as outlined in the CDC's
General Recommendation on Immunization (51(RR02) pp 1-36) Cancer is
diagnosed for example by physical exam, biopsy, blood test, or
x-ray.
[0161] The subject is e.g., any mammal, e.g., a human, a primate,
mouse, rat, dog, cat, cow, horse, pig, a fish or a bird. The
treatment is administered prior to diagnosis of the infection.
Alternatively, treatment is administered after diagnosis.
[0162] Efficaciousness of treatment is determined in association
with any known method for diagnosing or treating the particular
disorder or infection. Alleviation of one or more symptoms of the
disorder indicates that the compound confers a clinical
benefit.
Evaluation of Antigenic Protein Fragments (APFs) for Vaccine
Potential
[0163] A vaccine candidate targeting humoral immunity must fulfill
at least three criteria to be successful: it must provoke a strong
antibody response ("immunogenicity"); a significant fraction of the
antibodies it provokes must cross-react with the pathogen
("immunogenic fitness"); and the antibodies it provokes must be
protective. While immunogenicity can often be enhanced using
adjuvants or carriers, immunogenic fitness and the ability to
induce protection (as evidenced by neutralization) are intrinsic
properties of an antigen which will ultimately determine the
success of that antigen as a vaccine component.
Evaluation of Immunogenic Fitness
[0164] "Immunogenic fitness" is defined as the fraction of
antibodies induced by an antigen that cross-react with the
pathogen. (See Matthews et al., J. Immunol. 169:837 (2002)). It is
distinct from immunogenicity, which is gauged by the titer of all
of the antibodies induced by an antigen, including those antibodies
that do not cross-react with the pathogen. Inadequate immunogenic
fitness has probably contributed to the disappointing track record
of peptide vaccines to date. Peptides that bind with high affinity
to antibodies and provoke high antibody titers frequently lack
adequate immunogenic fitness, and, therefore, they fail as
potential vaccine components. Therefore, it is important to include
immunogenic fitness as one of the criteria for selecting influenza
vaccine candidates.
[0165] A common explanation for poor immunogenic fitness is the
conformational flexibility of most short peptides. Specifically, a
flexible peptide may bind well to antibodies from patients, and
elicit substantial antibody titers in naive subjects. However, if
the peptide has a large repertoire of conformations, a
preponderance of the antibodies it induces in naive subjects may
fail to cross-react with the corresponding native epitope on intact
pathogen.
[0166] Like short peptides, some APFs may be highly flexible and,
therefore may fail as vaccine components. The most immunogenically
fit APFs are likely to consist of self-folding protein subdomains
that are intrinsically constrained outside the context of the whole
protein.
[0167] Because immunogenic fitness is primarily a property of the
APF itself, and not of the responding immune system, immunogenic
fitness can be evaluated in an animal model (e.g. in mice) even
though ultimately the APF will have to perform in humans.
[0168] The immunogenic fitness achieved by APFs is evaluated by
immunosorption of anti-APF sera with purified spike or membrane
protein, in a procedure analogous to that described in Matthews et
al., J. Immunol. 169:837 (2002). IgG is purified from sera
collected from mice that have been immunized. Purified,
biotinylated proteins (as appropriate, depending on the particular
APF with which the mice were immunized) are mixed with the mouse
IgG and incubated. Streptavidin-coated sepharose beads are then
added in sufficient quantity to capture all of the biotinylated
protein, along with any bound IgG. The streptavidin-coated beads
are removed by centrifugation at 13,000 rpm in a microcentrifuge,
leaving IgG that has been depleted of antibodies directed against
the protein, respectively. Mock immunoabsorptions are performed in
parallel in the same way, except that biotinylated BSA will be
substituted for influenza protein as a mock absorbent.
[0169] To measure the immunogenic fitness of APFs, the absorbed
antibodies and the mock-absorbed antibodies are titered
side-by-side in ELISA against the immunizing APF. For APFs affinity
selected from a phage display NPL, the antigen for these ELISAs
will be purified APF-GST fusion proteins. For the potentially
glycosylated APFs from the mammalian cell display NPL, the antigen
for these ELISAs will be APF-Fc fusion proteins secreted by
mammalian cells and purified with protein A. The percentage
decrease in the anti-APF titer of absorbed antibodies compared with
the mock-absorbed antibodies will provide a measure of the
immunogenic fitness of the APF.
Methods of Treatment
[0170] The invention provides for both prophylactic and therapeutic
methods of treating a subject at risk of (or susceptible to) an
influenza virus-related disease or disorder. Such diseases or
disorders include but are not limited to, e.g., bird flu.
Prophylactic Methods
[0171] In one aspect, the invention provides methods for preventing
an influenza virus-related disease or disorder in a subject by
administering to the subject a monoclonal antibody or scFv antibody
of the invention or an agent identified according to the methods of
the invention. For example, scFv and/or monoclonal antibody D7, D8,
F10, G17, H40, A66, D80, E88, E90, and H98 may be administered in
therapeutically effective amounts. Optionally, two or more
anti-influenza antibodies are co-administered
[0172] Subjects at risk for an influenza virus-related diseases or
disorders include patients who have come into contact with an
infected person or who have been exposed to the influenza virus in
some other way. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the
influenza virus-related disease or disorder, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression.
[0173] The appropriate agent can be determined based on screening
assays described herein. Alternatively, or in addition, the agent
to be administered is a scFv or monoclonal antibody that
neutralizes an influenza virus that has been identified according
to the methods of the invention.
[0174] Therapeutic Methods
[0175] Another aspect of the invention pertains to methods of
treating an influenza virus-related disease or disorder in a
patient. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein and/or an scFv antibody or monoclonal antibody identified
according to the methods of the invention), or combination of
agents that neutralize the influenza to a patient suffering from
the disease or disorder.
[0176] Combinatory Methods
[0177] The invention provides treating an influenza-related disease
or disorder, such as bird flu, in a patient by administering two or
more antibodies, such as D7, D8, F10, G17, H40, A66, D80, E88, E90,
and H98 that bind to the same epitope of the HA protein.
[0178] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
General Methods
Expression and Preparation of Various Panning Antigens for
Selection of Phage Display Antibody Library.
[0179] HA 1.
[0180] HA 1 is the N-terminal fragment (aa17-338) of (A/Thailand/2
(SP-33)/2004 (H5N1). The gene was codon-optimized and expressed as
fusion protein with a C-terminal 9 amino-acids tag (C9-tag:
GTETSQVAPA (SEQ ID NO: 103)). The fusion protein HA1-C9 was
expressed in 293T cells transiently and the secreted protein was
purified from supernatant by affinity chromatography. Protein A
Sepharose covalently coupled with anti-C9 antibody 1D4 (National
Cell Culture Center) was used for purification of HA1-C9.
[0181] Trimeric HA0.
[0182] The ectodomain (HA0) of hamagglutinin (HA) gene of A/Viet
Nam/1203/2004 was expressed in insect cells as a fusion protein by
adapting the protocol described previously.sup.33. This construct
contains a C-terminal trimerizing `foldon` sequence from the
bacteriophage T4 fibritin to stabilize the trimeric structure,
followed by a thrombin site and a His6 tag. The cDNA of the fusion
protein was cloned into the baculovirus transfer vector, pAcGP67A
(BD Biosciences, Bedford, Mass.) to allow for efficient secretion
of recombinant protein. 9.times.10.sup.6 cells were infected with
the viral stock. 3 days after infection, the cells were spun down
and the supernatant was incubated with 6 ml Ni-NTA beads (Qiagen
Inc., Valencia, Calif.). The beads were washed with TBS buffer (10
mM Tris.HCl/80 mM NaCl, pH8.0) with 10 mM imidazole and eluted with
TBS with 250 mM Imidazole. The eluted HAS protein was dialyzed
against TBS buffer and further purified by ion-exchange using Mono
Q HR10/10 column (GE Healthcare, Piscataway, N.J.). The purified
HAS were digested by thrombin overnight. The integrity and property
of HA0 trimer was examined using Gel filtration (Superdex 200
column) and SDS-PAGE.
Selection of Phage Antibody Libraries and Screening of Antibodies
Against H5.
[0183] Two human non-immune scFv libraries (a total of
2.7.times.10.sup.10 members) constructed from B-cells of 57
un-immunized donors were used for selection of scFvs against the
purified HA1 or trimeric HA0. 5.times.10.sup.11 pfu of phage-scFvs
prepared from each library were incubated with immunotubes (Nunc,
Naperville, Ill.) coated with 10 .mu.g of HA1 or HA0, separately.
The selection procedures were the same as described previously.
After two rounds of selection, randomly picked single phage-scFv
clones were screened for specific binding to HA1 or HA0 by
enzyme-linked immunosorbent assay (ELISA) as described previously.
Clones that bound to HA1 or HA0 with A.sub.450 values of >1.0
were scored as positive, whereas negative clones gave values of
<0.1. For HA1 or HA0 specific binding clones, the genes of
variable regions of heavy (VH) and light (VL) chain were sequenced
and their corresponding amino acid sequences were aligned to
identify antibodies with different sequence for further
characterization.
Expression and Purification of Soluble scFv-Fcs and Full-Length
Human IgG1.
[0184] Phage-scFvs of individual clones were produced for
neutralization assay using the same method as making phage library.
Phage particles were concentrated 25 times by using PEG/NaCl
precipitation. scFv-Fcs and whole human IgG1s were produced as
described previously. In brief, selected scFvs were converted to
scFv-Fcs by subcloning the scFv into a Fc expression vector pcDNA
3.1-Hinge which contains the hinge, CH2, and CH3 domains of human
IgG1 but lacks CH1. For whole human IgG1s, the VH and VL gene
fragments of scFv were separately subcloned into human IgG1 kappa
light chain or lambda light chain expression vector TCAE5 or TCAE6.
scFv-Fcs or IgG1 s were expressed in 293T or 293F cells
(Invitrogen) by transient transfection and purified by protein A
sepharose affinity chromatography.
Surface Plasmon Resonance (SPR) Analysis
[0185] Kinetic analyses of H5 HA Mabs binding to recombinant HA0
(VietNam1203/04) trimer were performed on a Biacore T100 (Biacore)
at 25.degree. C. Anti-human IgG Fc antibody (Biacore) was
covalently coated to individual flow cell surfaces of a CM4 sensor
chip by amine-coupling using the amine coupling kit (Biacore). HA
Mabs were captured onto anti-human IgG Fc surfaces at the flow rate
of 10 ul/min in HBS buffer (Biacore) to ensure that the Mab-HA0
binding occurred as a homogenous 1:1 Langmuir interaction. HA0 was
injected over each flow cell at the flow rate of 30 ul/min in HBS
buffer, and at concentrations ranging from to 0.31 to 20 nM. A
buffer injection served as a negative control. All experiments
contained an additional anti-human IgG Fc antibody control surface
that served to account for changes in the buffer refractive index
and to test for potential nonspecific interactions between HA0 and
anti-human IgG Fc. Upon completion of each association and
dissociation cycle, surfaces were regenerated with 3M MgCl.sub.2
solution. The association rates (ka), dissociation rate constants
(kd), and affinity constants (KD) were calculated using Biacore
T100 evaluation software. The goodness of each fit was based on the
agreement between experimental data and the calculated fits, where
the Chi.sup.2 values were below 1.0. Surface densities of Mabs
against HA0 were optimized to minimize mass transfer and avoid any
contribution of avidity effects. All ka, kd, KD reported here
represent the means and standard errors of three experiments.
Viruses and Cells
[0186] Wild type influenza A/Vietnam/1203/2004 (H5N1; H5-VN04),
A/HongKong/483/1997 (H5N1; H5-HK97), A/Netherlands/219/2003 (H7N7;
H7-NL03), and A/Ohio/4/1983 (H1N1; H1-OH83) viruses as well as a
cold-adapted vaccine strain of A/Ann Arbor/6/1960 (H2N2) were
obtained from the WHO Global Influenza Surveillance Network and
provided by Alexander Klimov (CDC, Atlanta, USA). H1-PR34 and
Madin-Darby canine kidney (MDCK) cells were obtained from the
American Type Culture Collection and propagated in Dulbecco's
Modification of Eagle's Medium with 10% fetal bovine serum. Viral
infectivity was determined by plaque assay on MDCK cells. Live
wildtype H5N1 viruses were handled in biosafety level 3
containment, including enhancements required by the U.S. Department
of Agriculture and the Select Agents program
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
Neutralization Assay with HA Pseudotyped Viruses
Plasmids and Constructs
[0187] The full length HA gene of A/Thailand/2(SP-33)/2004(H5N1)),
H5-SP33, and neuramidase gene of A/Vietnam 1203/2004, N1, were
codon-optimized and cloned into pcDNA3.1 vector to obtain the
pcDNA3.1-H5-SP33 and pcDNA3.1-N1 expression plasmid, separately.
The pCAGGS-H1(SC) plasmid encoding A/South Carolina/1/1918 (H1N1)
full length HA protein was kindly provided by form Dr. P. Palese
(Mount Sinai School of Medicine, US). The pCAGGS-H1(PR) plasmid
encoding A/Puerto Rico/8/34 (H1N1) HA protein was a generous gift
from Dr. M. Farzan (Harvard Medical School, US).
Making the HA-Pseudotyped Viruses.
[0188] The single-round HIV luciferase reporter viruses of H5N1 or
H1N1 pseudotyped by H5-SP33, H1-SC/1918 or H1-PR/34 were made by
co-transfection of 293T cells with 4 plasmids that are
pcDNA3.1-H5-SP33, HIV packaging vector pCMV.DELTA.R 8.2 encoding
HIV-1 Gag-Pol.sup.57, transfer vector pHIV-Luc encoding the firefly
luciferase reporter gene under control of the HIV-1 LTR, and
expressing plasmid pcDNA3.1-N1. 36 hours post-transfection, viral
supernatants were harvested and stored at 4.degree. C.
Neutralization assay.
[0189] Before transducing cells, viral supernatant of H1-SC/1918 or
H1-PR/34F pseudotyped viruses was incubated with 16 .mu.g/mL
TPCK-treated trpsin for one hour at 25.degree. C. and then
neutralized with trypsin neutralizing solution (TNS, Cambrex) at
the ratio of 1:1 (V/V). Testing antibodies or sera were incubated
with adequate amount of H5-SP33 pseudotyped or trpsin-treated H1
pseudotyped viruses for 30 mins at RT. The mixture was then added
to 293T cells in 96 well plates and continued the culture for 48
hours. Viral entry level was evaluated by measuring the luciferase
activity in the target cells with a microplate luminometer.
Epitope Mapping of Antibody 8, 10 and 66
[0190] Plasmids and Constructs.
[0191] pcDNA3.1-H5-SP33, pCAGGS-H1(SC) and pCAGGS-H1(PR) were
described as above. pCAGGS-H7 (FPV) which encodes HA proteins for
A/FPV/Rostock/34 (H7N1) was kindly provided by Dr. X. Yang (Beth
Israel Deaconess Medical Center). All the mutants of
pcDNA3.1-H5-SP33 or pCAGGS-H7 (FPV) were constructed by the
QuikChange method (Stratagene). Flow cytometry analysis of the
binding of anti-H5 antibodies to HA expressing cells. Various
full-length wild type HA and HA mutants expressing plasmids for H1,
H5 or H7 were transfected transiently into 293T cells. 48 hours
after transfection, anti-H5 antibodies (10 ug/ml) were incubated
with transfected 293T cells at 4.degree. C. for 1 hour. Cells were
then washed three times with PBS containing 0.5% BSA and 0.1% NaN3.
For the detection of anti-H5 antibodies' binding to HA transfected
cells, FITC-labeled goat anti-human IgG (Pierce) was used as
secondary antibody and incubated with cells at 4.degree. C. for 30
min Cells were washed again as above and analyzed using a Becton
Dickinson FACScaliber with CellQuest software.
Microneutralization Assay
[0192] The method was performed as described previously.sup.48.
Briefly, 100 TCID.sub.50 (median tissue culture infectious doses)
of virus were mixed in equal volume with log 2 dilutions of
antibody stock solution (1 mg/ml) in 96-well tissue culture plates,
and incubated for 1 h at 37.degree. C. Indicator MDCK cells
(1.5.times.10.sup.4 cells/well) were added to the plates, followed
by incubation at 37.degree. C. for 20 hours. To establish the
endpoint, cell monolayers were then washed with PBS, fixed in
acetone, and viral antigen detected by indirect ELISA with a mAb
against influenza A NP (A-3, Accurate).
Plaque Reduction Assay
[0193] A/Viet Nam 1203/2004 (H5N1) or A/Netherland/219/03 (H7N7)
viruses (10,000 pfu) were incubated with anti-H5 scFv-Fcs at three
different concentrations 100 ug/ml, 10 ug/ml and 1 ug/ml at
37.degree. C. for 30 mins The virus-antibody mixture was
transferred onto MDCK cell monolayers in 12-well plates and
incubated at 37.degree. C. for 1 h and cells were then washed and
overlaid with agar. After 4 days of incubation, the agar overlay
was discarded, and plaques were visualized by crystal violet
staining.
Hemagglutination Inhibition (HI) Assay
[0194] Avian influenza viruses preferentially bind to sialic acid
receptors that contain N-acteylneuraminic acid .alpha.2,3-galactose
(.alpha.2,3Gal) linkages while human viruses preferentially bind to
those containing N-acetylneuaminic acid .alpha.2,6-galactose
((.alpha.2,6Gal) linkages. The molecular basis for receptor
specificity is most likely determined by a complex set of residues
in the viral HA. Hemagglutination-inhibition (HI) tests are a
simple and widely used method to assess antibody responses to
influenza hemagglutinin Stephenson et al. reported that by using
horse erythrocytes and thereby increasing the proportion of
.alpha.2,3 Gal linkages available for binding, the sensitivity of
the hemagglutination assay was improved. In addition, the titers of
the HI assay correlated well with the titers of the
micro-neutralization assay. The pseudotyped viruses will be first
titered using hemagglutination of horse erythrocytes as described.
The initial high-throughput screens will be similarly performed
using horse erythrocyte suspensions and assaying for the ability of
the phage antibodies to inhibit the HI endpoint of the titered
H5/N1 viruses. More detailed secondary screens for inhibition of
H5N1 reporter virus entry and HI will be preformed using dose
response studies with soluble scFvFc proteins produced by
transiently transfected 293T cells and purified by protein A
beads.
Viral Binding Inhibition Assay
[0195] 0.5.times.10.sup.6 293T cells were incubated with
H5-TH04-pseudotyped HIV viruses (.about.500 ng of p24) in the
presence of anti-H5 mAbs, control mAbs, or in the absence of
antibodies, in a buffer of PBS containing 0.5% BSA and 0.02%
NaN.sub.3 at 4.degree. C. After an hour of incubation, cells were
spun down. Supernatants were collected and tested for p24 levels
using a HIV-1 p24.sup.CA capture ELISA kit (NCI, Frederick, NIH) to
quantify the unbound viruses. The cells were then washed one or two
times and lysed to quantify the cell-bound virus using the same
method.
Cell Fusion Inhibition Assay
[0196] 293T cells, .about.90% confluent in six-well plates, were
transfected with pcDNA3.1-H5-TH04 plasmid and pcDNA 3.1-N1 at a
ratio of 4:1 (3 .mu.g total DNA/well) using lipofectamine 2000
(Invitrogen). The culture medium was supplemented with 1 ml of
anti-H5, control or mock mAbs at 6 hours post-transfection, and
cells cultured for 36 hours. Cells were observed for syncytia
formation with a phase-contrast microscope. Photomicrographs were
taken at 10.times. magnification.
Expression and Preparation of Various HA Proteins for Panning
[0197] HA1 is an N-terminal fragment of HA of H5N1
A/Thailand/2(SP-33)/2004 (H5-TH04), residues 11 to 325 (H3
numbering). The gene was codon-optimized and expressed as fusion
protein with a C-terminal 9 amino-acids tag (C9-tag: GTETSQVAPA).
The fusion protein HA1-C9 was expressed in 293T cells transiently
and the secreted proteins in supernatant were harvested 48 hours
after transfection and purified from the supernatant by affinity
chromatography using Protein A Sepharose that coupled covalently
with anti-C9 antibody 1D4 (National Cell Culture Center). The
method to produce HA0 protein of H5-VN04 is the same as described
below for crystallization of H5-F10 complex but without the
baculovirus coinfection for furin-cleavage.
[0198] ELISA
[0199] 0.2 .mu.g of pure H5 HA proteins was coated onto 96-well
Maxisorb ELISA plate (Nunc, NY) at 2 .mu.g/mL in PBS at 4.degree.
C. overnight. The plate was washed with PBS for 3 times to remove
uncoated proteins. For regular ELISA, 1 .mu.g/mL of anti-H5
scFv-Fcs followed by HRP-anti-human IgG1 were used to detect the
binding of anti-H5 scFv-Fcs to H5 HA proteins. For competition
ELISA, 50 .mu.L (10.sup.12 pfu) of anti-H5 phage-scFvs were mixed
with 5 .mu.g/mL of anti-H5 scFv-Fcs and applied to H5-VN04 HA
coated ELISA plate. The competition of scFv-Fcs for the binding of
phage-scFvs to HA0 were determined by measuring the remaining
binding of phage-scFvs using HRP-anti-M13. The optical density at
450 nm was measured after incubation of peroxidase
tetramethylbenzidine (TMB) substrate system (KPL, Gaithersburg
Md.).
Expression, Purification, and Crystallization of the H5-F10
Complex
[0200] The gene encoding single chain (VH-linker-VL) F10 (scFv) was
cloned into pSynI vector containing an N-terminal periplasmic
secretion signal pelB, and a C-terminal 6.times.His tag. F10 scFv
was expressed in XL10 cells in 2YT media containing 0.1% glucose
(w/v) at 25.degree. C. for 15 hours with 0.5 mM IPTG. Protein was
purified first by Hisbind Ni-NTA (Novagen) according to the
manufacturer's instructions, and then by Superdex 200 (Amersham
Biosciences) in 50 mM Tris-HCl, 0.5 M NaCl, pH 8. The ectodomain of
H5-VN04 HA gene was expressed in insect cells as a fusion protein
by adapting the protocol described previously.sup.6. This construct
contains a C-terminal trimerizing `foldon` sequence from the
bacteriophage T4 fibritin to stabilize the trimeric structure,
followed by a thrombin site and a His.sub.6 tag. The cDNA of the
fusion protein was cloned into the baculovirus transfer vector,
pAcGP67A (BD Biosciences, Bedford, Mass.), to allow for efficient
secretion of recombinant protein. To obtain fully cleaved HA (as
HA1-HA2 trimers), sf9 cells were co-infected with baculovirus
stocks of HA0 and furin at an empirically derived ratio. The furin
cDNA was a gift from Dr. Robert Fuller (University of Michigan).
Three days after infection, the cells were spun down and the
supernatant was incubated with Ni-NTA beads (Qiagen Inc., Valencia,
Calif.). The beads were washed with TBS buffer (10 mM Tris.HCl, 80
mM NaCl, pH8.0) with 10 mM imidazole, and eluted with TBS with 250
mM Imidazole. The eluted H5 protein was dialyzed against TBS buffer
and further purified by ion-exchange using Mono Q HR10/10 column
(GE Healthcare, Piscataway, N.J.). The purified H5 was digested by
thrombin overnight and further purified by Superdex 200 column in
TBS buffer.
[0201] H5-F10 complexes were formed by mixing the two purified
components, and isolated by Superdex 200 in TB S buffer. Peak
fractions were pooled and concentrated to .about.11 mg/ml. The
integrity of the H5 trimer was examined using Gel filtration
(Superdex 200 column) and SDS-PAGE. Crystals grew by the hanging
drop vapor diffusion method at 22.degree. C. Two .mu.L of H5-F10
were mixed with an equal volume of 12.5% PEG 1K, 25% ethylene
glycol, 100 mM Tris, pH 8.5. Crystals were flash-frozen in liquid
nitrogen prior to data collection.
Co-Crystallization of the H5-F10 Complex
[0202] For co-crystallization of the H5-F10 complex, F10 was
expressed in E. coli and purified by Ni-NTA and gel filtration. H5
was expressed by co-infection of H5 and furin baculovirus stocks in
sf9 cells, and purified by Ni-NTA, anion ion exchange, and gel
filtration chromatography. H5-F10 complexes were obtained by mixing
H5 with an excess of F10 and isolated by gel filtration. Crystals
were grown by mixing 2 .quadrature.L of complex (.about.11 mg/ml)
with an equal volume of 12.5% PEG 1K, 25% ethylene glycol, 100 mM
Tris, pH 8.5 using the hanging drop vapor diffusion method at
22.degree. C. The crystal structure was determined at 3.2 .ANG.
resolution by Molecular Replacement and refined to an R.sub.FREE of
0.29 with excellent geometry.
Data Collection, Structure Determination, and Refinement
[0203] X-ray diffraction data were collected at the Stanford
Synchrotron Radiation Laboratory (SSRL) beam-lines 7.1 and 9.2.
Data were processed with XDS.sup.7 and the HKL2000 package.
[0204] The structure of the H5-F10 complex was determined by
molecular replacement with PHASER using the structure of H5
(A/Vietnam/1194/04; PDB code 2IBX) and the scFv structure of SARS
nAb 80R (PDB code 2 GHW) as search models. The scFv structure
homology model was build with WHATIF.sup.9. The asymmetric unit
contains two H5 trimers and six F10 molecules.
[0205] Solutions from molecular replacement were subjected to
several rounds of refinement with the program REFMAC5 with
simulated annealing in CNS and manual model rebuilding with Coot
and Xtalview. The final model includes 506/503/503/496/495/496
residues for 6 independent copies of HA, respectively, 233 residues
for each nAb10, 24 N-acetyl-d-glucosamine and 6.beta.-d-mannose, 0
water molecules. Geometric parameters were assessed with PROCHECK
and Rampage.
Protection of Mice with hMabs Against H5
[0206] All mouse studies were performed in USDA and CDC accredited
biosafety level 3 (BSL3) animal facility in accordance with
protocols approved by the CDC Animal Care and Use Committee. Female
8-10 weeks old Balb/C mice (5 per group) were used in all
experiments. For all groups, mice were observed for 2 weeks,
weighted and deaths noted daily.
[0207] Prophylactic Mouse Study.
[0208] Three hMabs (D8-IgG1, F10-IgG1 and A66-IgG1) or control hMab
80R-IgG1 at 2 doses of 10 mg/kg and 2.5 mg/kg.sup.53 were
administered into mice intraperitoneally (i.p.). 24 hours after
hMabs administration, all groups of mice were challenged with
A/Vietnam/1203/04(H5N1) or A/HK/483/97 (H5N1) intranasally (i.n.)
at dose of 10 MLD.sub.50 (50% mouse lethal dose) viruses under
light anesthesia.
[0209] Therapeutic Mouse Study.
[0210] Mice were first infected i.n. with 10 MLD.sub.50 of
A/Vietnam/1203/04(H5N1) or A/HK/483/97 (H5N1). Three hMabs against
H5 or control hMab at 10 mg/kg were then i.p. administered into
mice 24, 48 and 72 hours after A/Vietnam/1203/04(H5N1) infection.
For A/HK/483/97 (H5N1) infected mice, hMabs were only injected 24
hours after viral challenge.
Identification of Anti-H5 Phage Antibodies
[0211] Antibodies Against HA1.
[0212] Purified recombinant HA1 was used to select antibodies from
two non-immune human scFv libraries separately. After two rounds of
selection on HA1, 58 out of 96 clones screened by ELISA were HA1
specific positive clones. Three unique anti-HA1 scFvs were
identified (38B and 1C) by sequencing analysis of the 58
HA1-positive clones. Similar selection and screening experiments
were repeated; no new clones were identified but obtained the same
three scFvs as described above.
[0213] Antibodies Against Ectodomain of H5 (HA0).
[0214] Purified recombinant trimeric HA0 was used to select the
same antibody libraries as that used for HA1. After two rounds of
selection on HA0, a total of 392 clones were screened for HA0
specific binding by ELISA. 97 clones recognized HA0 protein
specifically. Ten unique anti-HA0 scFvs (7, 8, 10, 17, 40, 66, 80,
88, 90 and 98) were identified by sequence analysis. Six different
VH and 10 different VL genes were revealed (some scFvs shared the
same VH gene). Five out of the six different VH belong to one gene
family, IGHV1-69. The VL genes were much diverse than the VH genes,
three out of the 10 VL are Kappa chain. (FIG. 3).
Establishment of a Reliable Single-Round Reporter Virus System
[0215] The single-round HIV luciferase reporter viruses of H5N1 or
H1N1 pseudotyped by H5-SP33, H1-SC/1918 or H1-PR/34 were made by
co-transfection of 293T cells with 4 plasmids that are
pcDNA3.1-H5-SP33, HIV packaging vector pCMV.DELTA.R 8.2 encoding
HIV-1 Gag-Pol, transfer vector pHIV-Luc encoding the firefly
luciferase reporter gene under control of the HIV-1 LTR, and
expressing plasmid pcDNA3.1-N1. 36 hours post-transfection, viral
supernatants were harvested and stored at 4.degree. C. The titer of
HA0-pseudotyped viruses was measured by infecting 293T cells.
Including the N1 expressing plasmid in the transfection of making
pseudotyped viruses can dramatically increase the titer of
H5-pseudotyped viruses The proper behavior of the H5-pseudotyped
viruses was examined by using ferret immune serum against H5N1
(A/VietNam/2004). These viruses can be neutralized potently by
anti-serum, while not by pre-bleeding serum. Thus a reliable
single-round high efficient reporter virus system was established
for efficient screening of neutralizing antibody
Example 2
Identification of Neutralizing Antibodies Against H5N1 Using
Viruses Pseudotyped with 115-SP33 and Microneutralization Assay
Results of Neutralization Assay Using Pseudotyped Viruses.
[0216] Bivalent scFvFcs of anti-H5 antibodies were produced and
tested for neutralization activities against HA0-pseudotyped
viruses. We found that 2A is a moderate neutralizing antibody, 38B
and 1C are non-neutralizing antibodies (data not shown). The 2A
antibody has been used as a useful anti-HA reagent for other assays
(epitope mapping) described below.) By using HA0 trimer as a
target, we have identified 10 new unique antibodies against HA0.
These Abs were screened for neutralization activity by using a
novel high-throughput phage-scFvs screening method that has been
recently set up in our lab. Briefly, the phage-scFvs of individual
clones were directly used in a neutralization assay immediately
following being screened by ELISA for positive clones in 96-well
plates. In this schema, neutralization activity will be known in 48
hours. By doing this, we bypass steps of subcloning and expressing
of soluble Abs which are time-consuming.
[0217] As shown in the figure, neutralizing Abs that were indicated
by phage turned out to be potent neutralizing when confirmed with
soluble scFvFc Abs. All the 10 antibodies screened by phage-scFvs
are potent neutralizing Abs. We also converted three of the 10
antibodies (D8, F10 and A66) into full-length human IgG1, the
potent neutralization activity were again seen with H5-SP33
pseudotyped viruses. The three antibodies also cross-neutralized
H1N1: potently neutralized stain of H1-SC/1918 and moderately
neutralized strain of H1-PR/34 (FIGS. 4A-4C).
[0218] Results of microneutralization assay. As shown in FIGS.
4A-4C and 9B, the 10 anti-H5 antibodies exhibited different levels
of neutralization. F10-Fc, A66-Fc, E88-Fc and E90-Fc appeared to be
the most potent inhibitors, which neutralize more than 95% of
10,000 pfu virus at 10 ug/ml and more than 50% at 1 ug/ml. We also
have seen complete plaque reduction when 100 pfu was used for these
four antibodies at 10 ug/ml or 1 ug/ml (data not shown).
[0219] The 10 antibodies (scFv-Fcs) were found to neutralize H5
pseudo-viruses (virus-like particles with HIV-1 only cores that
display H5 on their surface), in this case from the Glade 1 virus,
A/Thailand/2-SP-33/2004 (H5N1) ("H5-TH04"). All 10 antibodies also
exhibited high but distinct levels of neutralization against
H5-VN04 (Clade 1) and A/Indonesia/5/2005 ("H5-1N05", Clade 2.1)
viruses in a stringent plaque reduction assay, suggesting that the
neutralization epitope(s) is conserved across different H5 clades.
However, none of the Abs neutralized a Group 2 virus, HPAI H7N7
strain, A/Netherlands/219/03 (H7N7) (H7-NL03) (FIG. 4).
[0220] All 10 nAbs bound to trimeric H5 and cross-competed against
each other (FIG. 9), indicating that they recognize an overlapping
epitope. Based on this finding as well as VH sequence diversity and
neutralization potency, three nAbs (D8, F10 and A66) were selected
and converted into full-length human IgG1. All three nAb IgG1s
bound to recombinant H5 (H5-VN04) in vitro with high affinity
(Kd.about.100-200 pM) and very slow dissociation rates
(kd.about.10.sup.-4s.sup.-1) (FIG. 10).
Example 3
Kinetic Characterization of the Binding of HA0 (the Ectodomain of
HA of A/Vietnam1203/04) to D8-IgG1, F10-IgG1 and A66-IgG1 by using
BIAcore T-100
[0221] Mabs were captured on a CM4 chip via anti-human IgG1, and
trimeric HA0 at various concentrations (20, 10, 5, 2.5, 2.5, 1.25,
0.625 nM) was injected over the chip surface. Binding kinetics was
evaluated using a 1:1 Langmuir binding model. The recorded binding
curves (with blank reference subtracted) are shown in black and the
calculated curves in red. All ka, kd, KD represent the means and
standard errors of three experiments. Using Biacore T100 and
Biacore T100 evaluation software, we determined the kinetic rates
and affinity constants for the three anti-HA MAbs by fitting to a
1:1 Langmuir model. As shown in the figure below, similar ka, kd,
and KD of D8 and F10 were observed, and A66 had a 1.8 fold lower KD
than 8 and 10 due to a relative faster dissociation rate than the
other two antibodies. The complexes between Mabs and HA0 were
stable as illustrated by a very slow dissociation rate
(8.8.+-.1.1.times.10.sup.-5s.sup.-1.about.1.8.+-.0.3.times.10
S.sup.-1) and high affinity binding with H5 trimer at pM level.
(FIG. 10)
Example 4
Prophylactic and Therapeutic Effect of Anti-H5 Antibodies
[0222] The protective efficacy of human nAbs against H5N1 and H1N1
virus infection was evaluated in a BALB/c mouse model. Mice were
treated with different doses of nAb either before or after lethal
viral challenge.
[0223] Prophylactic Efficacy (FIGS. 7 a, b, g and h).
Mice were treated with anti-H5 nAbs or control mAb 24 hour before
lethal challenge by intranasally (i.n.) with 10 median lethal doses
(MLD.sub.50) of the H5N1 or H1N1s. Intra-peritoneal (i.p.)
injection of 10 mg/kg of any of the three nAbs provided complete
protection of mice challenged with H5-VN04 (A/Vietnam/1203/04
(H5N1), Clade 1). A lower antibody dose (2.5 mg/kg) was also highly
protective. (FIG. 7a) Prophylactic protection against H5-HK97
(A/HongKong/483/97 (H5N1), Clade 0) virus was observed in 80-100%
of the mice treated with 10 mg/kg of any of the three nAbs. (FIG.
7b) Any of the three nAbs (at 10 mg/kg of single injection)
provided complete protection of mice challenged with H1-WSN33
(A/WSN/1933(H1N1)) viruses. (FIG. 7g) D8 and F10 completely
protected mice challenged with H1-PR34 (A/Puerto Rico/8/34 (H1N1))
when given at 10 mg/kg of single injection. A66 provided complete
protection of mice when 25 mg/kg of antibody was given as a single
injection. (FIG. 7h)
[0224] Therapeutic Efficacy (FIG. 7c-f).
[0225] Mice were inoculated with H5-VN04 and injected with nAbs at
24, 48, 72 hpi (FIGS. 7 c, e and f) or with H5-HK97 at 24 hpi (FIG.
7 d). I.p. treatment with 15 mg/kg (a therapeutically achievable
dose in humans) of any of the 3 nAbs at 24 h post-inoculation (hpi)
protected 80-100% of mice challenged with 10-times the MLD.sub.50
of either H5-VN04 or H5-HK97 virus (FIG. 7c-d). Mice treated with
the same dose of nAbs at 48 or 72 hpi with H5-VN04 showed similar
or higher levels of protection (FIG. 7 2e-f). Surviving mice in all
anti-H5 nAb treatment groups remained healthy and showed minimal
body weight loss over the 2-week observation period (data not
shown).
Example 5
Suppression of Viral Titer in Tissues by Anti-115 Antibodies in a
Therapeutic Animal Study
[0226] The anti-viral effect of nAbs used therapeutically was
further investigated by measuring virus titer 4 days post-challenge
either in lungs, as an indicator of inhibition of local
replication, or in spleen and brain, indicative of the systemic
spread that is characteristic of H5N1 infection. The nAbs mediated
a significant suppression of viral replication in lungs of H5 nAb
treated mice as compared to controls when given within 48 hours of
challenge (FIG. 12). Notably, two of the nAbs, D8 and F10, also
showed antiviral effect when given at 72 hpi. Systemic virus spread
to the brain was low in control animals, obscuring any effect of
nAb treatment. However, the impact of nAb therapy on systemic
spread was dramatically demonstrated by >1000-fold suppression
of virus spread to the spleen even when the three nAbs were given
within 72 hpi.
Example 6
Initial Epitope Mapping of Antibody D8, F10 and A66
[0227] Various full-length wild type HA and HA mutants expressing
plasmids for H5-SP33 were transfected transiently into 293T cells.
48 hours after transfection, anti-H5 antibodies (10 ug/ml) were
incubated with transfected 293T cells at 4.degree. C. for 1 hour.
Cells were then washed three times with PBS containing 0.5% BSA and
0.1% NaN3. For the detection of anti-H5 antibodies' binding to HA
transfected cells, FITC-labeled goat anti-human IgG (Pierce) was
used as secondary antibody and incubated with cells at 4.degree. C.
for 30 min. Antibody 2A was used as a control antibody to indicate
the expression of each mutant. As we can see from the table below,
the epitopes for Mab D8, 1F0 and A66 are similar and they are
located at positions of 307 on HA1 and 52, 59, 65, 93 on HA2. The
position of these amino acids were highlighted in the crystal
structure of H5 (A/Vietnam/1203/04(H5N1))
Example 7
Binding and Neutralization of Cluster H1 Viruses
[0228] The cross-clade neutralization of H5N1 viruses (FIG. 4)
prompted testing for neutralization of other HA subtypes within the
Group 1 H1 cluster (FIG. 11). All three nAbs bound to cells
expressing full-length H1 ((A/South Carolina/1/1918 (H1N1)
("H1-SC1918") and A/Puerto Rico/8/34 (H1N1) ("H1-PR34")), H2
(A/Japan/305/57(H2N2)) ("H2-JP57"), H6 (A/chicken/New
York/14677-13/1998(H6N2)) (H6-NY98), but not a Group 2 subtype, H7
((A/FPV/Rostock/34 (H7N1) ("H7-FP34")) (FIG. 4). They also
neutralized H1-SC1918- and H1-PR34-pseudotyped virus infection
(FIG. 4); and in addition, F10 neutralized PR34 (H1N1), A/Ohio/83
(H1N1) ("H1-OH83") and A/Ann Arbor/6/60 (H2N2) infection (FIG. 4).
These results suggest that these nAbs recognize an epitope on HA
that is not only highly conserved among H5 clades, but is also
present on H1, H2 and H6 subtypes, and potentially subtypes within
other clusters. We therefore decided to investigate the mechanism
of virus neutralization in detail.
Example 8
nAbs Inhibit Cell Fusion but not Virus Binding
[0229] Two ways in which anti-HA Abs can mediate neutralization are
blocking HA binding to its cellular receptor, and inhibition of
virus-host membrane fusion by interfering with low-pH induced
conformational changes of HA. We found that the three nAbs neither
affected virus binding to cells nor inhibited haemagglutination
(binding and cross-linking of red blood cells), while a mouse
anti-H5 nAb, 17A2.1.2, and ferret anti-H5N1 serum, both of which
inhibit haemagglutination, reduced binding to background
levels.
[0230] We next tested the ability of the nAbs to inhibit fusion of
host cell membranes using a model system in which 293T cells at
high density were co-transfected with H5- and N1-expressing
plasmids from H5-TH04. Surface-expressed viral proteins cause cells
to fuse and form syncytia, which occurs spontaneously within 36-48
hrs post-transfection under normal culture conditions. When nAbs
were added 6 hrs post-transfection and observed 36 hrs later,
complete inhibition of syncytia formation was observed in all 3
cases In the absence of nAbs, cells exposed briefly (3-4 minutes)
to an acidic medium (pH 5) 30 hrs post-transfection form extensive
syncytia. The nAbs (5 .mu.g/ml) also completely blocked syncytia
formation under these conditions (data not shown). Together, these
results indicate that all three nAbs inhibit membrane fusion
without interfering with receptor binding.
Example 9
Structural Characterization of the nAb Epitope
[0231] We next determined the epitope and mode of binding of one of
the nAbs, F10, by solving the crystal structure of its scFv
fragment in complex with HA (H5-VN04) at 3.2 .ANG. resolution, and
by mutagenesis. (FIG. 6 and Table 4)
[0232] In the complex, each H5 trimer binds three molecules of F10,
at symmetry-related sites, burying .about.1500 .ANG..sup.2 of
protein surface per antibody; the structure of H5 itself is not
significantly altered by F10 binding. HA is synthesized as a single
chain, HA0, that is activated by proteolytic cleavage into two
subunits, HA1 and HA2. Cleavage leads to the burial of the "fusion
peptide" (comprising the first .about.21 residues of HA2) into the
membrane-proximal stem. F10 binding occurs exclusively in this
region (FIG. 6), making intimate contacts with the fusion peptide,
elements of HA1 and HA2 (both of which are integral to the
structure of this region) that lock the peptide into place in the
neutral pH conformation, as well as the large helical hairpin of
HA2 that undergoes a massive conformation change at acidic pH in
order to propel the fusion peptide from its viral membrane-proximal
pocket to the distal surface of the virus, where it can trigger
fusion with the endosomal membrane.
[0233] The heavy chain of F10 plays the major role in H5 binding,
utilizing the tips of its three complementarity-determining regions
(CDRs). Each F10 molecule make contacts with both the HA1 and HA2
subunits within a single monomer of the HA trimer (FIG. 6). The
contact region comprises a pocket formed by part of the HA2 fusion
peptide, with elements of HA1 on one side and an exposed face of
helix .alpha.A of HA2 on the other (FIG. 6). A triad of antibody
residues--F55 and M54 from CDR H2, and Y102 from CDR H3--form major
contact points. The phenyl ring of F55 lies across a flat surface
formed by a prominent loop of the fusion peptide loop (HA2 residues
DGW 19.sub.2-21.sub.2 (H3 numbering scheme; subscripts 1 and 2
refer to HA1 and HA2 chains)) and the aromatic side-chains of two
flanking histidines (residues 18.sub.1 and 38.sub.1) and a
tryptophan, W21.sub.2, which forms the back of the pocket. The
side-chain of M54 also contacts the aromatic rings of W21.sub.2 and
H38.sub.1, as well as the side-chain of I45.sub.2 from helix
.alpha.A, while its main-chain carbonyl oxygen hydrogen-bonds with
the side-chain of H38.sub.1. Y102 inserts its side-chain into a
hydrophobic crevice created by four side-chains of the .alpha.A
helix, and also hydrogen-bonds to a backbone carbonyl (D19.sub.2)
of the fusion peptide. The CDR H1 loop makes multiple contacts with
the C-terminal end of helix .alpha.A and a loop of HA1 at the base
of the head region (FIG. 6).
[0234] In parallel, mutagenesis experiments were carried out on
helix .alpha.A to help define the epitope (FIG. 6). Mutations in 3
residues that directly contact the antibody: V52A/E, N53A and 156A,
significantly reduced or ablated antibody binding, while the
conservative mutation, V52L, had no effect. As controls, mutations
on a different exposed face of the helix, which does not contact
the antibody, had no effect on antibody binding. Thus, mutagenesis
of the .alpha.A helix is fully consistent with the epitope defined
crystallographically for F10. Furthermore, the other two nAbs, for
which no structural data exist, showed an almost identical
mutant-nAb binding profile, indicative of a closely overlapping
epitope and consistent with competitive binding (FIG. 6, FIG. 9).
Taken together, we conclude that all three nAbs neutralize virus by
stabilizing the neutral pH conformation of HA in a region that
provides the trigger (most likely release of the fusion peptide
from its pocket) for conformational changes that lead to the
fusogenic state.
Example 10
Structural Basis of Broad-Spectrum Neutralization
[0235] The region of HA defined by the F10 epitope is highly
conserved among the 16 HA subtypes (FIG. 6 and Table 5). The fusion
peptide must adopt a well-defined structure, both in the neutral pH
conformation and during the process of membrane fusion, and
mutagenesis studies have shown that few sequence changes are
tolerated. The exposed helical face of HA2 .alpha.A is also nearly
invariant across both groups, presumably because it must reorganize
to form the inner surface of the long trimeric coiled-coil in the
fusogenic state. Thus, the requirement of these segments to adopt
two very different structures in two distinct environments
presumably imparts strong evolutionary constraints on the
sequences, providing a clear rationale for their conservation
across the 16 HA subtypes.
[0236] Crystal structures have been determined for subtypes H1, H5,
and H9 from Group 1 and H3 and H7 from Group 2, and the structures
fall into two distinct classes, coincident with the two groups
defined by phylogenetic analysis. Further comparisons show that the
two classes are also readily distinguishable at the detailed
3-dimensional level within the region defined by the F10 epitope.
Sequence differences in a few key amino acids buried in the core
that are characteristic of each class (e.g. at 17.sub.1 and
111.sub.2) lead to consistent differences in the orientation of
several side-chains within the epitope, as well as the disposition
of the pocket with respect to helix .alpha.A.
[0237] Within each group/class, the epitope region is highly
conserved, with very few differences in a given cluster, and only a
few changes, generally conservative, between clusters (e.g., at
positions 18.sub.1 and 38.sub.1, FIG. 6D). Our observation that the
3 nAbs can recognize all 4 members of the H1 cluster demonstrates
that amino-acid differences at the periphery of the epitope (such
as at residue 40.sub.1, which is K, Q or V in this cluster) may not
be critical for binding.
[0238] The 10 antibodies we selected have 5 different sets of VH
CDR1-3, which vary substantially in sequence and length.
Nevertheless, the three key residues in CDR2 (M54, F55) and CDR3
(Y102) that insert into the pocket are conserved. The conservation
of key residues within a varied CDR context may expand the range of
HA subtype/cluster recognition by this group of nAbs beyond those
tested here. However, it should be noted that 4 out of 6 Group 2
subtypes are glycosylated at position 38.sub.1, which lies at the
periphery of the F10 epitope. Modeling studies predict steric
clashes (but only with the CDR H1 loop), which likely contributes
to the observed lack of binding/neutralization of H7 viruses.
Example 11
Anti-H5 nAbs Bind to a Broad Range of Group 1 HA Subtypes
[0239] Group 1 viruses contains 10 of the total 16 subtypes of
Influenza A virus, and is further classified into 3 "clusters",
H1a, H1b, and H9. We tested our nAbs for binding to all seven HA
subtypes of Clusters H1a and H1b, which include avian H5 as well as
the most common human influenza subtypes, with the exception of the
Group 2 subtype, H3. In addition to H5, we found that all three nAb
IgG1s bound to cells expressing full-length H1 from 2 different
strains of H1N1, including the 1918 "Spanish flu"; H2 from H2N2;
and H6 from H6N2; as well as the Cluster 1b subtypes: H11 from
H11N9; H13 from H13N6; and H16 from H16N3. However, none of them
bound to a Group 2 subtype, H7 from H7N1 (FIG. 13). More details
about FIG. 13 are as follows. FACS analysis of anti-H5 nAbs binding
to H1, H2, H5, H6 (cluster H1a) and H11, H13 and H16 (Cluster H1b).
293T cells were transiently transfected with different
HA-expressing plasmids. NAb binding to the cells was analyzed by
FACS. H5-specific antibody 2A and 80R are negative control. Lack of
binding to a Group 2 HA, H7, is also shown. Detail on viral strains
are: H1-SC1918 ((A/South Carolina/1/1918 (H1N1)), H1-PR34 (A/Puerto
Rico/8/34 (H1N1)), H2-JP57 (A/Japan/305/57(H2N2)), H5-TH04
(A/Thailand/2-SP-33/2004 (H5N1)), H6-NY98 (A/Chicken/New
York/14677-13/1998 (H6N2)), H7-FP34 (A/FPV/Rostock/34 (H7N1)),
H11-MP74 (A/Duck/memphis/546/74 (H11N9)), H13-MD77
(A/Gull/MD/704/77 (H13N6)) and H16-DE06 (A/Shorebird/DE/172/06
(H16N3)).
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TABLE-US-00021 [0287] TABLE 3 Contact residues at the H5-F10
interface FRH1 CDRH1 CDRH2 CDRH3 F10 S25 V27 T28 S30 S31 M54 F55
T57 P100 S101 Y102 I103 S105 HA1 S291 S291 Q40 38 H10 M292 H38 HA2
I56 V52 I45 W21 V18 V18 Q42 Q42 D19 D19 K38 N53 T49 I45 D19 K3 K38
G20 T41 W21 Q42 I45 Contact residues defined by having a closest
interatomic distance of <4.5 .ANG.. The color scheme indicates
contributions of individual residues to the binding free energy:
very favorable (red(V27, T28, I45, D19, W21)), favorable
(orange(S25, S291, M292, I56, V52, N53, T49, H18, V18, G20, T57,
T41)), negligible (blue (S30, S31, Q40, H38), unfavorable (black),
very unfavorable (bold black). Based on energy calculations using
the server at
http://structure.pitt.edu/servers/fastcontact.sup.1.
TABLE-US-00022 TABLE 4 Data collection and refinement statistics.
H5-F10 Data Collection Cell parameters a = 205.3, b = 118.5, c =
338.9 .beta. = 99.6.degree. Space group C2 Resolution (.ANG.)* 3.2
(3.28-3.20) Total reflections 509705 Unique reflections 112570
Completeness (%)* 85.0 (68.4) Average I/.sigma.(I)* 9.5 (2.0)
R.sub.MERGE (%)* 12.8 (81.0) Redundancy* 4.5 (4.5) .sigma. cutoff
-3 Refinement Resolution 50-3.2 (3.28-3.20) R.sub.WORK* 0.23 (0.32)
R.sub.FREE (5% data)* 0.29 (0.38) RMSD bond distance 0.01 (.ANG.)
RMSD bond angle (.degree.) 1.31 Average B value 75.7 Solvent atoms
0 .sigma. cutoff none Ramachandran plot Residues in favored 90.0
regions (%) Residues in allowed 9.5 regions (%) Residues in outlier
0.5 regions (%) *Numbers in parentheses correspond to the highest
resolution shell.
TABLE-US-00023 TABLE 5* Sequence comparison of F10 epitope among 16
HA subtypes. # Full-length # Unique HA1 HA2 Group Cluster Subtype
sequence sequence 17 18 38 40 291 18 Group 1 H1a H2 100 95 Y H H
(Q, E)/K T (I)/V (18)/77 (20)/75 H5 1620 1178 (S, T, F)/Y (Y, M)/H
(Q, Y)/H (K)/Q (I, N, T, R)/S (I)/V (4)/1174 (2)/1176 (4)/1174
(2)/1176 (6)/1172 (6)/1172 H1 1211 701 Y H H (I)V (S)/N (V, M)/I
(2)/699 (16)685 (137)/546 H6 278 230 Y H H V (I)N (V)/I (1)229
(43)/187 H1b H13 16 16 Y L S (V)/I N I (6)/16 H16 8 6 Y L S (I)/V N
I (2)/6 H11 64 64 Y L S (I)/V (T)/S (L)/I (1)/64 (1)/63 (1)/63 H9
H8 10 10 Y Q Q M S I H12 19 18 Y Q Q E S V H9 252 234 (H)/Y (L)/Q
(D)/H (R, E)/K (I)/T V (33)/201 (1)/233 (1)/233 (5)/229 (1)/233
Group 2 H3 H4 105 90 H H (A)T (R)Q (A, I, S, N)/T I (1)/89 (1)/89
(10)/82 H14 2 2 H H S K D I H3 2302 1228 H H N (N)/T (H, Y, E, G)/D
(M)/(V/I) (1)/1227 (35)/1193 (149)/(70%/30 H7 H15 8 5 H H N T P I
H7 334 273 H H N T (S, R, P)/N (V)/I (131)/142 (41)232 H10 31 28 H
H N T (E)/K V (1)/27 HA2 Group Cluster Subtype 19 20 21 38 41 42 45
Group 1 H1a H2 D G W K T Q (F, V)/I (39)/56 H5 (N, H, Y, X)/D G
(R)/W (Q, R, N)/K (S)/T P/Q (M, L, T)/I (5)/1173 (1)/1177 (23)/1155
(3)/1175 (1)/1177 (8)/1170 H1 D G W (K, R, L)/Q T Q (V)/I (60)/641
(2)/699 H6 D G (R)/W (R)/K T Q (V)/I (1)/229 (102)/128 (78)/152 H1b
H13 N G W K T Q I H16 N G W K T Q I H11 N G W (R)/K T Q (V)/I
(5)/59 (3)/61 H9 H8 D G W Q T Q I H12 A G W R T Q I H9 (S)/A G
(G)/W (K, G)/R T Q (V, F, M, R)/I (1)/233 (1)/233 (14)/220 (43)/191
Group 2 H3 H4 D G (G)/W L T Q I (1)/91 H14 D G W L T Q I H3 (N)/D G
W (F)/L T Q (V, T, L)/I (15)/1213 (1)/1227 (5)/1223 H7 H15 D G W Y
T Q I H7 (N)/D G W (H)/Y T (P)/Q (V)/I (57)/213 (1)/272 (2)/271
(3)/270 H10 (N, E)/D (A)/G W Y T Q I (2)/26 (1)/27 HA2 Group
Cluster Subtype 49 52 53 56 111 Group 1 H1a H2 T (I)/V N I H (1)/94
H5 (I)/T V N (F)/V H (1)/1177 (1)/1177 H1 (S, N, X)/T (M, I)/V N
(V)/I H (19)/682 (2)/699 (1)/700 H6 (I)/T (I)/V N I H (1)/229
(3)/227 H1b H13 T V N I H H16 T V N I H H11 (I)/T V N (I, A)/V H
(1)/63 (17)/47 H9 H8 T (I)/V N I H (1)/9 H12 Q L N I H H9 (I)/T V
(S, T, D)/N (I)/V (C)/H (1)/233 (3)/231 (55)/179 1/233 Group 2 H3
H4 (N)/T L N I (A)/T (1)/89 (2)/90 H14 N L N I T H3 (D, S, T, A)/N
(M)/L (D)/N (V, T, F)/I (A)/T (22)/1206 (1)/1227 (1)/1227 (19)/1209
(7)/1221 H7 H15 T L N I A H7 T L N I (T)/A (35)/238 H10 T L N (V)/I
A (1)/27 *This table represents sequences available in public
influenza sequence databases
(http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) as of Apr. 17,
2008. Light blue highlights: top `( )/`, (amino acid
variant(s))/amino acid consensus at the position; bottom `( )/`,
(number of amino acid variants)/number of consensus amino acids.
Non-highlighted amino acids are 100% conserved or variants are
observed .ltoreq.5 times at those positions for subtypes H4, H6,
H9, H10, H11. Histidines H17 (HA1) and H111 (HA2) that may play a
role in pH-trigger are highlighted in green. Table 5 discloses
"INGW" as SEQ ID NO: 105, "IDGW" as SEQ ID NO: 106, and "VAGW" as
SEQ ID NO: 107.
Other Embodiments
[0288] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. Other aspects, advantages, and modifications considered
to be within the scope of the following claims. The claims
presented are representative of the inventions disclosed herein.
Other, unclaimed inventions are also contemplated. Applicants
reserve the right to pursue such inventions in later claim.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 172 <210> SEQ ID NO 1 <211> LENGTH: 369
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 1 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagt
gacaatgcta tcagctgggt gcgacaggcc 120 ccaggacaag ggcttgagtg
gatggggggc atcattccta tctttggaaa accaaactac 180 gcacagaagt
tccagggcag agtcacgatt actgcggacg aatccacgag cacagcctac 240
atggacctga ggagcctgag atctgaggac acggccgttt attactgtgc gagagattca
300 gacgcgtatt actatggttc ggggggtatg gacgtctggg gccaaggcac
cctggtcacc 360 gtctcctca 369 <210> SEQ ID NO 2 <211>
LENGTH: 123 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 2 Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Asp Asn 20 25 30 Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile
Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ser Asp Ala Tyr Tyr Tyr Gly Ser Gly Gly
Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 3 <211> LENGTH: 335 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
3 ctgcctgtgc tgactcaatc atcctctgcc tctgcttccc tgggatcctc ggtcaagctc
60 acctgcactc tgagcagtgg gcatagtaac tacatcatcg catggcatca
acagcagcca 120 gggaaggccc ctcggtactt gatgaaggtt aatagtgatg
gcagccacac caagggggac 180 gggatccctg atcgcttctc aggctccagc
tctggggctg accgctacct caccatctcc 240 aacctccagt ctgaggatga
ggctagttat ttctgtgaga cctgggacac taagattcat 300 gtcttcggaa
ctgggaccaa ggtctccgtc ctcag 335 <210> SEQ ID NO 4 <211>
LENGTH: 111 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 4 Leu Pro Val Leu Thr Gln Ser Ser Ser
Ala Ser Ala Ser Leu Gly Ser 1 5 10 15 Ser Val Lys Leu Thr Cys Thr
Leu Ser Ser Gly His Ser Asn Tyr Ile 20 25 30 Ile Ala Trp His Gln
Gln Gln Pro Gly Lys Ala Pro Arg Tyr Leu Met 35 40 45 Lys Val Asn
Ser Asp Gly Ser His Thr Lys Gly Asp Gly Ile Pro Asp 50 55 60 Arg
Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr Ile Ser 65 70
75 80 Asn Leu Gln Ser Glu Asp Glu Ala Ser Tyr Phe Cys Glu Thr Trp
Asp 85 90 95 Thr Lys Ile His Val Phe Gly Thr Gly Thr Lys Val Ser
Val Leu 100 105 110 <210> SEQ ID NO 5 <211> LENGTH: 363
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg ctcctggagg tatcttcaac
accaatgctt tcagctgggt ccgacaggcc 120 cctggacaag gtcttgagtg
ggtgggaggg gtcatccctt tgtttcgaac agcaagctac 180 gcacagaacg
tccagggcag agtcaccatt accgcggacg aatccacgaa cacagcctac 240
atggagctta ccagcctgag atctgcggac acggccgtgt attactgtgc gagaagtagt
300 ggttaccatt ttaggagtca ctttgactcc tggggcctgg gaaccctggt
caccgtctcc 360 tca 363 <210> SEQ ID NO 6 <211> LENGTH:
121 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Pro
Gly Gly Ile Phe Asn Thr Asn 20 25 30 Ala Phe Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Gly Val Ile Pro
Leu Phe Arg Thr Ala Ser Tyr Ala Gln Asn Val 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Thr Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Ser Gly Tyr His Phe Arg Ser His Phe Asp Ser Trp Gly
100 105 110 Leu Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 7 <211> LENGTH: 363 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7
caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60 tcctgcaagg ctcctggagg tatcttcaac accaatgctt tcagctgggt
ccgacaggcc 120 cctggacaag gtcttgagtg ggtgggaggg gtcatccctt
tgtttcgaac agcaagctac 180 gcacagaacg tccagggcag agtcaccatt
accgcggacg aatccacgaa cacagcctac 240 atggagctta ccagcctgag
atctgcggac acggccgtgt attactgtgc gagaagtagt 300 ggttaccatt
ttaggagtca ctttgactcc tggggcctgg gaaccctggt caccgtctcc 360 tca 363
<210> SEQ ID NO 8 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Asn
Phe Met Leu Thr Gln Pro His Ser Val Ser Ala Ser Pro Gly Lys 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Asn Ile Ala Ala Asn
20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr
Thr Val 35 40 45 Ile Tyr Glu Asp Asp Arg Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala
Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp
Tyr Tyr Cys Gln Thr Tyr Asp Thr 85 90 95 Asn Asn His Ala Val Phe
Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 9 <211> LENGTH: 333 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 9 aattttatgc
tgactcagcc ccactctgtg tcggcgtctc cggggaagac ggtgaccatc 60
tcctgcaccg gcagcagtgg caacattgcc gccaactatg tgcagtggta ccaacaacgt
120 ccgggcagtg cccccactac tgtgatctat gaggatgacc gaagaccctc
tggggtccct 180 gatcggttct ctggctccat cgacaggtcc tccaactctg
cctccctcac catctcagga 240 ctgaagactg aggacgaggc tgactactac
tgtcagactt atgataccaa caatcatgct 300 gtgttcggag gaggcaccca
cctgaccgtc ctc 333 <210> SEQ ID NO 10 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala
Ser Gly Lys His Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Gly Thr Ser Asn Ile Gly Arg Asn 20 25 30 His Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn
Glu Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Val Ser Gly Leu Gln 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Asn Leu 85
90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 11 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 11 tcctatgagc tgactcagcc accctcagcg
tctgggaaac acgggcagag ggtcaccatc 60 tcttgttctg gaggcacctc
caacatcgga cgtaatcatg ttaactggta ccagcaactc 120 ccaggaacgg
cccccaaact cctcatctat agtaatgaac agcggccctc aggggtccct 180
gaccgattct ctggctccaa atctggcacc tccgcctccc tggccgtgag tgggctccag
240 tctgaggatg aggctgatta ttactgtgca tcatgggatg acaacttgag
tggttgggtg 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> SEQ
ID NO 12 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 12 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Tyr 20 25 30 Ala
Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Thr Gly Met Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Tyr Tyr Tyr Glu Ser Ser
Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 13 <211> LENGTH: 361
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg
caccttcagc gcttatgctt tcacctgggt gcggcaggcc 120 cctggacaag
ggcttgagtg gatgggaggc atcaccggaa tgtttggcac agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aactcacgag cacagcctac
240 atggagttga gctccctgac atctgaagac acggcccttt attattgtgc
gagaggattg 300 tattactatg agagtagtct tgactattgg ggccagggaa
ccctggtcac cgtctcctca 360 g 361 <210> SEQ ID NO 14
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 14 Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Cys Ser Tyr Ala
Gly His 85 90 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
Val Leu 100 105 110 <210> SEQ ID NO 15 <211> LENGTH:
361 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 15 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaggg cttctggagg
caccttcagc gcttatgctt tcacctgggt gcggcaggcc 120 cctggacaag
ggcttgagtg gatgggaggc atcaccggaa tgtttggcac agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aactcacgag cacagcctac
240 atggagttga gctccctgac atctgaagac acggcccttt attattgtgc
gagaggattg 300 tattactatg agagtagtct tgactattgg ggccagggaa
ccctggtcac cgtctcctca 360 g 361 <210> SEQ ID NO 16
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 16 Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Lys 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Ser Cys Gln Gln Tyr Asp Gly
Val Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Thr Val Glu Ile Lys
100 105 <210> SEQ ID NO 17 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 17 cagtctgtgc tgactcagcc accctccgcg
tccgggtctc ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcag
tgacgttggt ggttataact ctgtctcctg gtaccaacag 120 cacccaggca
aagcccccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtc 180
cctgatcgct tctctgcctc caagtctggc aacacggcct ccctgaccgt ctctgggctc
240 caggctgagg atgaggctga ttatttctgc tgctcatatg caggccacag
tgcttatgtc 300 ttcggaactg ggaccaaggt caccgtcctg 330 <210> SEQ
ID NO 18 <211> LENGTH: 124 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Thr Ser Ser Glu Val Thr Phe Ser Ser Phe 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40
45 Gly Gly Ile Ser Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Gln Ser Thr Arg Thr
Ala Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Ser Tyr Ile Cys Ser Gly
Gly Thr Cys Val Phe Asp 100 105 110 His Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 <210> SEQ ID NO 19 <211>
LENGTH: 324 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 19 gaaattgtgc tgactcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca
gagtcttagc agcaagtact tagcctggta tcagcagaaa 120 cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcaccc tcaccatcag tagactggag
240 cctgaagatt ttgcagtgta ttcctgtcag cagtatgatg gcgtacctcg
gacgttcggc 300 caagggacca cggtggaaat caaa 324 <210> SEQ ID NO
20 <211> LENGTH: 110 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 20 Gln Pro Gly Leu Thr
Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr
Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30 Gly
Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40
45 Ser Tyr Arg Asn Asn Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser
50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly
Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Thr Trp
Asp Ser Ser Leu 85 90 95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 <210> SEQ ID NO 21 <211>
LENGTH: 372 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 21 caggtgcagc tggtgcagtc aggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcacgt cctctgaagt
caccttcagt agttttgcta tcagctgggt gcgacaggcc 120 cctggacaag
ggcttgagtg gctgggaggg atcagcccta tgtttggaac acctaattac 180
gcgcagaagt tccaaggcag agtcaccatt accgcggacc agtccacgag gacagcctac
240 atggacctga ggagcctgag atctgaggac acggccgtgt attattgtgc
gagatctcct 300 tcttacattt gttctggtgg aacctgcgtc tttgaccatt
ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> SEQ ID NO
22 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 22 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 <210> SEQ ID NO 23 <211> LENGTH: 372
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 23 caggtacagc tgcagcagtc aggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcacgt cctctgaagt
caccttcagt agttttgcta tcagctgggt gcgacaggcc 120 cctggacaag
ggcttgagtg gctgggaggg atcagcccta tgtttggaac acctaattac 180
gcgcagaagt tccaaggcag agtcaccatt accgcggacc agtccacgag gacagcctac
240 atggacctga ggagcctgag atctgaggac acggccgtgt attattgtgc
gagatctcct 300 tcttacattt gttctggtgg aacctgcgtc tttgaccatt
ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> SEQ ID NO
24 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 24 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Val Thr Phe Ser Ser Tyr 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ile Gly Val Phe Gly Val Pro Lys Tyr Ala Gln Asn Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Pro Thr Ser Thr
Val Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Gly Tyr Tyr Val Gly Lys
Asn Gly Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 25 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 cagcctgggc tgactcagcc accctcggtg
tccaagggct tgagacagac cgccacactc 60 acctgcactg ggaacagcaa
caatgttggc aaccaaggag cagcttggct gcagcagcac 120 cagggccacc
ctcccaaact cctatcctac aggaataatg accggccctc agggatctca 180
gagagattct ctgcatccag gtcaggaaac acagcctccc tgaccattac tggactccag
240 cctgaggacg aggctgacta ttactgctca acatgggaca gcagcctcag
tgctgtggta 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> SEQ
ID NO 26 <211> LENGTH: 110 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 26 Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Ile
Leu Thr Cys Thr Gly Asp Ser Asn Asn Val Gly His Gln 20 25 30 Gly
Thr Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40
45 Ser Tyr Arg Asn Gly Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser
50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ile Gly
Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp
Asp Ser Ser Leu 85 90 95 Ser Ala Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 <210> SEQ ID NO 27 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 27 gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca
gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc
ctaagctcct gatctatgct gcatccagtt tgcaaagagg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagac ttcactctca ccattagcag cctgcagcct
240 gaagattttg cagtgtatta ctgtcagcag tatgatagtt caccgtacac
ttttggccag 300 gggaccaagg tagagatcaa a 321 <210> SEQ ID NO 28
<211> LENGTH: 126 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 28 Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Arg Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Val Trp Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr
65 70 75 80 Met Glu Val Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp
Gln Pro Glu Ala 100 105 110 Leu Asp Ile Trp Gly Leu Gly Thr Thr Val
Thr Val Ser Ser 115 120 125 <210> SEQ ID NO 29 <211>
LENGTH: 366 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 29 caggtgcagc tggtgcaatc tggggctgaa
gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaaga cttctggagt
caccttcagc agctatgcta tcagttgggt gcgacaggcc 120 cctggacaag
ggcttgagtg gatgggaggg atcatcggtg tctttggtgt accaaagtac 180
gcgcagaact tccagggcag agtcacaatt accgcggaca aaccgacgag tacagtctac
240 atggagctga acagcctgag agctgaggac acggccgtgt attactgtgc
gagagagccc 300 gggtactacg taggaaagaa tggttttgat gtctggggcc
aagggacaat ggtcaccgtc 360 tcttca 366 <210> SEQ ID NO 30
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 30 Gln Pro Val Leu Thr Gln Pro
Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Pro
Cys Gly Gly Asn Asn Ile Gly Gly Tyr Ser Val 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Leu Leu Val Ile Tyr 35 40 45 Asp
Asp Lys Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ala 50 55
60 Asn Ser Gly Ser Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80 Asp Glu Gly Asp Tyr Tyr Cys Gln Val Trp Asp Ser Gly Asn
Asp Arg 85 90 95 Pro Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 <210> SEQ ID NO 31 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 tcctatgagc tgactcagcc accctcggtg
tccaagggct tgagacagac cgccatactc 60 acctgcactg gagacagcaa
caatgttggc caccaaggta cagcttggct gcaacaacac 120 cagggccacc
ctcccaaact cctatcctac aggaatggca accggccctc agggatctca 180
gagagattct ctgcatccag gtcaggaaat acagcctccc tgaccattat tggactccag
240 cctgaggacg aggctgacta ctactgctca gtatgggaca gcagcctcag
tgcctgggtg 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> SEQ
ID NO 32 <211> LENGTH: 123 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 32 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Pro Phe Ser Met Thr 20 25 30 Ala
Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr
Ala Asn 65 70 75 80 Met Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn
Asn Asp Ala Phe Ala Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 33 <211> LENGTH:
378 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 33 caggtgcagc tggtgcagtc tggggctgag
gtgaggaagc ctggggcctc agtgaaggtc 60 tcatgtaagg cttctggata
caccttcacc ggttattata ttcactgggt gcgacaggcc 120 cctggacaag
gacttgagtg gatgggttgg atcaacccta tgactggtgg cacaaactat 180
gcacagaagt ttcaggtctg ggtcaccatg acccgggaca cgtccatcaa cacagcctac
240 atggaggtga gcaggctgac atctgacgac acggccgtgt attactgtgc
gaggggggct 300 tccgtattac gatattttga ctggcagccc gaggctcttg
atatctgggg cctcgggacc 360 acggtcaccg tctcctca 378 <210> SEQ
ID NO 34 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 34 Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly
Ser Ser Pro Gln 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 <210> SEQ ID NO 35 <211> LENGTH: 324
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 35 cagcctgtgc tgactcagcc accctcggtg
tcagtggccc caggacagac ggccagcatt 60 ccctgtgggg ggaacaacat
tggaggctac agtgtacact ggtaccaaca aaagccgggc 120 caggcccccc
tcttggtcat ttatgacgat aaagaccggc cctcagggat ccctgagcga 180
ttctctggcg ccaactctgg gagcacggcc accctgacaa tcagcagggt cgaagccggg
240 gatgagggcg actactactg tcaggtgtgg gatagtggta atgatcgtcc
gctgttcggc 300 ggagggacca agctgaccgt ccta 324 <210> SEQ ID NO
36 <211> LENGTH: 123 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 36 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Pro Phe Ser Met Thr 20 25 30 Ala
Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr
Ala Asn 65 70 75 80 Met Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn
Asn Asp Ala Phe Ala Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 37 <211> LENGTH:
369 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37 caggtgcagc tggtgcagtc tggggctgaa
gtgaagaagc ctggctcctc ggtgaaggtt 60 tcctgcaagg cttctggagg
ccccttcagc atgactgctt tcacctggct gcgacaggcc 120 cctggacaag
ggcttgagtg gatgggtggg atcagcccta tctttcgtac accgaagtac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagccaac
240 atggagctga ccagcctgaa atctgaggac acggccgtgt attactgtgc
gagaaccctt 300 tcctcctacc aaccgaataa tgatgctttt gctatctggg
gccaagggac aatggtcacc 360 gtctcttca 369 <210> SEQ ID NO 38
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 38 Leu Pro Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Arg
65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
Arg Leu 85 90 95 Ser Ala Ser Leu Phe Gly Thr Gly Thr Thr Val Thr
Val Leu 100 105 110 <210> SEQ ID NO 39 <211> LENGTH:
318 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 39 gaaattgtgt tgacgcagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct
240 gaagattttg cagtctattt ctgtcagcag tatggtagct cacctcaatt
cggccaaggg 300 acacgactgg agattaaa 318 <210> SEQ ID NO 40
<211> LENGTH: 369 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 40 caggtgcagc tggtgcagtc
tggggctgaa gtgaagaagc ctggctcctc ggtgaaggtt 60 tcctgcaagg
cttctggagg ccccttcagc atgactgctt tcacctggct gcgacaggcc 120
cctggacaag ggcttgagtg gatgggtggg atcagcccta tctttcgtac accgaagtac
180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa
cacagccaac 240 atggagctga ccagcctgaa atctgaggac acggccgtgt
attactgtgc gagaaccctt 300 tcctcctacc aaccgaataa tgatgctttt
gctatctggg gccaagggac aatggtcacc 360 gtctcttca 369 <210> SEQ
ID NO 41 <400> SEQUENCE: 41 000 <210> SEQ ID NO 42
<211> LENGTH: 330 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 42 ctgcctgtgc tgactcagcc
accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg
gaagcagctc caacatcgga agtaatactg taaactggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct
180 gaccgattct ctggctccag gtcaggcacc tcagcctccc tggccatcat
tggactccgg 240 cctgaggatg aagctgatta ttactgtcag tcgtatgaca
gcaggctcag tgcttctctc 300 ttcggaactg ggaccacggt caccgtcctc 330
<210> SEQ ID NO 43 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Consensus Sequence <400>
SEQUENCE: 43 Ser Tyr Ala Phe Ser 1 5 <210> SEQ ID NO 44
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 44 Thr Asn Ala Phe Ser 1 5
<210> SEQ ID NO 45 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 Ala
Tyr Ala Phe Thr 1 5 <210> SEQ ID NO 46 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 46 Ser Phe Ala Ile Ser 1 5 <210> SEQ ID
NO 47 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 47 Ser Tyr Ala Ile Ser
1 5 <210> SEQ ID NO 48 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
48 Gly Tyr Tyr Ile His 1 5 <210> SEQ ID NO 49 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 49 Met Thr Ala Phe Thr 1 5 <210> SEQ ID
NO 50 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 50 Asp Asn Ala Ile Ser
1 5 <210> SEQ ID NO 51 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Consensus Sequence
<400> SEQUENCE: 51 Gly Ile Ile Pro Met Phe Gly Thr Pro Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> SEQ ID NO 52
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 52 Gly Val Ile Pro Leu Phe Arg
Thr Ala Ser Tyr Ala Gln Asn Val Gln 1 5 10 15 Gly <210> SEQ
ID NO 53 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 53 Gly Ile Ile Gly Met
Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
<210> SEQ ID NO 54 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 54 Gly
Ile Ser Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10
15 Gly <210> SEQ ID NO 55 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
55 Gly Ile Ile Gly Val Phe Gly Val Pro Lys Tyr Ala Gln Lys Phe Gln
1 5 10 15 Gly <210> SEQ ID NO 56 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 56 Trp Ile Asn Pro Met Thr Gly Gly Thr Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Val <210> SEQ ID NO 57
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 57 Gly Ile Ser Pro Ile Phe Arg
Thr Pro Lys Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> SEQ
ID NO 58 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 58 Gly Ile Ile Pro Ile
Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
<210> SEQ ID NO 59 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Consensus Sequence <400>
SEQUENCE: 59 Ser Ser Gly Tyr Tyr Tyr Gly Gly Gly Phe Asp Val 1 5 10
<210> SEQ ID NO 60 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 60 Ser
Ser Gly Tyr His Phe Gly Arg Ser His Phe Asp Ser 1 5 10 <210>
SEQ ID NO 61 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 61 Gly Leu
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr 1 5 10 <210> SEQ ID NO 62
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 62 Ser Pro Ser Tyr Ile Cys Ser
Gly Gly Thr Cys Val Phe Asp His 1 5 10 15 <210> SEQ ID NO 63
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 63 Glu Pro Gly Tyr Tyr Val Gly
Lys Asn Gly Phe Asp Val 1 5 10 <210> SEQ ID NO 64 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 64 Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp
Gln Pro Glu Ala Leu Asp 1 5 10 15 Ile <210> SEQ ID NO 65
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 65 Thr Leu Ser Ser Tyr Gln Pro
Asn Asn Asp Ala Phe Ala Ile 1 5 10 <210> SEQ ID NO 66
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 66 Asp Ser Asp Ala Tyr Tyr Tyr
Gly Ser Gly Gly Met Asp Val 1 5 10 <210> SEQ ID NO 67
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 67 Thr Gly Ser Ser Ser Asn Ile
Gly Asn Tyr Val Ala 1 5 10 <210> SEQ ID NO 68 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 68 Thr Gly Ser Ser Ser Asn Ile Ala Ala Asn
Tyr Val Gln 1 5 10 <210> SEQ ID NO 69 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
Sequence <400> SEQUENCE: 69 Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr Asn Ser Val Ser 1 5 10 <210> SEQ ID NO 70 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 70 Thr Gly Asn Ser Asn Asn Val Gly Asn Gln
Gly Ala Ala 1 5 10 <210> SEQ ID NO 71 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 71 Thr Gly Asp Ser Asn Asn Val Gly His Gln
Gly Thr Ala 1 5 10 <210> SEQ ID NO 72 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 Gly Gly Asn Asn Ile Gly Gly Tyr Ser Val
His 1 5 10 <210> SEQ ID NO 73 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 73 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
Ala 1 5 10 <210> SEQ ID NO 74 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Arg Ala Ser Gln Ser Leu Ser Ser Lys Tyr
Leu Ala 1 5 10 <210> SEQ ID NO 75 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 75 Thr Gly Ser Ser Ser Asn Ile Gly Asn Tyr
Val Ala 1 5 10 <210> SEQ ID NO 76 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 76 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
Thr Val Asn 1 5 10 <210> SEQ ID NO 77 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 77 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu
Asn 1 5 10 <210> SEQ ID NO 78 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 78 Thr Leu Ser Ser Gly His Ser Asn Tyr Ile
Ile Ala 1 5 10 <210> SEQ ID NO 79 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
Sequence <400> SEQUENCE: 79 Ser Asn Ser Asp Arg Pro Ser 1 5
<210> SEQ ID NO 80 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 80 Glu
Asp Asp Arg Arg Pro Ser 1 5 <210> SEQ ID NO 81 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Glu Val Thr Lys Arg Pro Ser 1 5
<210> SEQ ID NO 82 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 Arg
Asn Asn Asp Arg Pro Ser 1 5 <210> SEQ ID NO 83 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 83 Arg Asn Gly Asn Arg Pro Ser 1 5
<210> SEQ ID NO 84 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 84 Asp
Asp Lys Asp Arg Pro Ser 1 5 <210> SEQ ID NO 85 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 85 Asp Ala Ser Asn Arg Ala Thr 1 5
<210> SEQ ID NO 86 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 86 Gly
Ala Ser Ser Arg Ala Thr 1 5 <210> SEQ ID NO 87 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Ser Asn Asn Gln Arg Pro Ser 1 5
<210> SEQ ID NO 88 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 88 Ala
Ala Ser Ser Leu Gln Arg 1 5 <210> SEQ ID NO 89 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 Ser Asn Glu Gln Arg Pro Ser 1 5
<210> SEQ ID NO 90 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 Val
Asn Ser Asp Gly Ser His Thr Lys Gly Asp 1 5 10 <210> SEQ ID
NO 91 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus Sequence <400> SEQUENCE: 91 Gln
Ser Tyr Asp Ser Leu Ser Ala Tyr Val 1 5 10 <210> SEQ ID NO 92
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 92 Gln Ser Tyr Asp Thr Asn Asn
His Ala Val 1 5 10 <210> SEQ ID NO 93 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Cys Ser Tyr Ala Gly His Ser Ala Tyr Val 1
5 10 <210> SEQ ID NO 94 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
94 Ser Thr Trp Asp Ser Ser Leu Ser Ala Val Val 1 5 10 <210>
SEQ ID NO 95 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 95 Ser Val
Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> SEQ ID NO 96
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 96 Gln Val Trp Asp Ser Gly Asn
Asp Arg Pro Leu 1 5 10 <210> SEQ ID NO 97 <211> LENGTH:
9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Gln Gln Tyr Gly Ser Ser Pro Gln Val 1 5
<210> SEQ ID NO 98 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Gln
Gln Tyr Asp Gly Val Pro Arg Thr 1 5 <210> SEQ ID NO 99
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 99 Gln Ser Tyr Asp Ser Arg Leu
Ser Ala Ser Leu 1 5 10 <210> SEQ ID NO 100 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 100 Gln Gln Tyr Asp Ser Ser Pro Tyr Thr 1 5
<210> SEQ ID NO 101 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 101
Ala Ser Trp Asp Asp Asn Leu Ser Gly Trp Val 1 5 10 <210> SEQ
ID NO 102 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 102 Glu Thr Trp Asp
Thr Lys Ile His Val 1 5 <210> SEQ ID NO 103 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide <400> SEQUENCE: 103 Gly Thr Glu Thr Ser Gln
Val Ala Pro Ala 1 5 10 <210> SEQ ID NO 104 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic 6xHis tag <400> SEQUENCE: 104 His His His His His
His 1 5 <210> SEQ ID NO 105 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Influenza A virus <400>
SEQUENCE: 105 Ile Asn Gly Trp 1 <210> SEQ ID NO 106
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Influenza A virus <400> SEQUENCE: 106 Ile Asp Gly Trp 1
<210> SEQ ID NO 107 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Influenza A virus <400> SEQUENCE:
107 Val Ala Gly Trp 1 <210> SEQ ID NO 108 <211> LENGTH:
4 <212> TYPE: PRT <213> ORGANISM: Influenza A virus
<400> SEQUENCE: 108 Val Asp Gly Trp 1 <210> SEQ ID NO
109 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Influenza A virus <400> SEQUENCE: 109 Ile Ala Gly
Trp 1 <210> SEQ ID NO 110 <211> LENGTH: 87 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
110 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
1 5 10 15 Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln 20 25 30 Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Met Phe
Gly Thr Pro Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Ala Asp Glu Ser 50 55 60 Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala
Arg 85 <210> SEQ ID NO 111 <211> LENGTH: 87 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
111 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
1 5 10 15 Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln 20 25 30 Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe
Gly Thr Pro Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Ala Asp Glu Ser 50 55 60 Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala
Arg 85 <210> SEQ ID NO 112 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 112 Lys Lys Pro Gly Ser Ser Val Lys Val Ser
Cys Thr Ser Ser Glu Val 1 5 10 15 Thr Phe Ser Ser Phe Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu Trp Leu Gly
Gly Ile Ser Pro Met Phe Gly Thr Pro Asn 35 40 45 Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Gln Ser 50 55 60 Thr Arg
Thr Ala Tyr Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr 65 70 75 80
Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Tyr Ile Cys Ser Gly Gly 85
90 95 Thr Cys Val Phe Asp His Trp Gly Gln Gly Thr Leu 100 105
<210> SEQ ID NO 113 <211> LENGTH: 104 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 113
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 1 5
10 15 Thr Phe Ser Ala Tyr Ala Phe Thr Trp Val Arg Gln Ala Pro Gly
Gln 20 25 30 Gly Leu Glu Trp Met Gly Gly Ile Ile Gly Met Phe Gly
Thr Ala Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Leu 50 55 60 Thr Ser Thr Ala Tyr Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr 65 70 75 80 Ala Leu Tyr Tyr Cys Ala Arg
Gly Leu Tyr Tyr Tyr Glu Ser Ser Phe 85 90 95 Asp Tyr Trp Gly Gln
Gly Thr Leu 100 <210> SEQ ID NO 114 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 114 Lys Lys Pro Gly Ser Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly 1 5 10 15 Pro Phe Ser Met Thr Ala Phe Thr
Trp Leu Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu Trp Met Gly
Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys 35 40 45 Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser 50 55 60 Thr Asn
Thr Ala Asn Met Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr 65 70 75 80
Ala Val Tyr Tyr Cys Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn Asn 85
90 95 Asp Ala Phe Ala Ile Trp Gly Gln Gly Thr Met 100 105
<210> SEQ ID NO 115 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 115
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Thr Ser Gly Val 1 5
10 15 Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln 20 25 30 Gly Leu Glu Trp Met Gly Gly Ile Ile Gly Val Phe Gly
Val Pro Lys 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile
Thr Ala Asp Lys Pro 50 55 60 Thr Ser Thr Val Tyr Met Glu Leu Asn
Ser Leu Arg Ala Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg
Glu Pro Gly Tyr Tyr Val Gly Lys Asn 85 90 95 Gly Phe Asp Val Trp
Gly Gln Gly Thr Met 100 105 <210> SEQ ID NO 116 <211>
LENGTH: 105 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 116 Lys Lys Pro Gly Ser Ser Val Lys
Val Ser Cys Lys Ala Pro Gly Gly 1 5 10 15 Ile Phe Asn Thr Asn Ala
Phe Ser Trp Val Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu Trp
Val Gly Gly Val Ile Pro Leu Phe Arg Thr Ala Ser 35 40 45 Tyr Ala
Gln Asn Val Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser 50 55 60
Thr Asn Thr Ala Tyr Met Glu Leu Thr Ser Leu Arg Ser Ala Asp Thr 65
70 75 80 Ala Val Tyr Tyr Cys Ala Arg Ser Ser Gly Tyr His Phe Arg
Ser His 85 90 95 Phe Asp Ser Trp Gly Leu Gly Thr Leu 100 105
<210> SEQ ID NO 117 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 117
Arg Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 1 5
10 15 Thr Phe Thr Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Gln 20 25 30 Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Met Thr Gly
Gly Thr Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Val Trp Val Thr Met
Thr Arg Asp Thr Ser 50 55 60 Ile Asn Thr Ala Tyr Met Glu Val Thr
Arg Leu Thr Ser Asp Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg
Gly Ala Ser Val Leu Arg Tyr Phe Asp 85 90 95 Trp Gln Pro Glu Ala
Leu Asp Ile Trp Gly Leu Gly Thr Thr 100 105 110 <210> SEQ ID
NO 118 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 118 Gly Gly Thr Phe
Ser Ser Tyr Ala 1 5 <210> SEQ ID NO 119 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5
<210> SEQ ID NO 120 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 120
Glu Val Thr Phe Ser Ser Phe Ala 1 5 <210> SEQ ID NO 121
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 121 Gly Gly Thr Phe Ser Ala Tyr
Ala 1 5 <210> SEQ ID NO 122 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
122 Gly Gly Pro Phe Ser Met Thr Ala 1 5 <210> SEQ ID NO 123
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 123 Gly Val Thr Phe Ser Ser Tyr
Ala 1 5 <210> SEQ ID NO 124 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
124 Gly Gly Ile Phe Asn Thr Asn Ala 1 5 <210> SEQ ID NO 125
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 125 Gly Tyr Thr Phe Thr Gly Tyr
Tyr 1 5 <210> SEQ ID NO 126 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
126 Ile Ile Pro Met Phe Gly Thr Pro 1 5 <210> SEQ ID NO 127
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 127 Ile Ile Pro Ile Phe Gly Thr
Pro 1 5 <210> SEQ ID NO 128 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
128 Ile Ser Pro Met Phe Gly Thr Pro 1 5 <210> SEQ ID NO 129
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 129 Ile Ile Gly Met Phe Gly Thr
Ala 1 5 <210> SEQ ID NO 130 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
130 Ile Ser Pro Ile Phe Arg Thr Pro 1 5 <210> SEQ ID NO 131
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 131 Ile Ile Gly Val Phe Gly Val
Pro 1 5 <210> SEQ ID NO 132 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
132 Val Ile Pro Leu Phe Arg Thr Ala 1 5 <210> SEQ ID NO 133
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 133 Ile Asn Pro Met Thr Gly Gly
Thr 1 5 <210> SEQ ID NO 134 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 134 Ala Arg Ser Pro Ser Tyr Ile Cys Ser Gly
Gly Thr Cys Val Phe Asp 1 5 10 15 His <210> SEQ ID NO 135
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 135 Ala Arg Gly Leu Tyr Tyr Tyr
Glu Ser Ser Phe Asp Tyr 1 5 10 <210> SEQ ID NO 136
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 136 Ala Arg Thr Leu Ser Ser Tyr
Gln Pro Asn Asn Asp Ala Phe Ala Ile 1 5 10 15 <210> SEQ ID NO
137 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 137 Ala Arg Glu Pro
Gly Tyr Tyr Val Gly Lys Asn Gly Phe Asp Val 1 5 10 15 <210>
SEQ ID NO 138 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 138 Ala
Arg Ser Ser Gly Tyr His Phe Arg Ser His Phe Asp Ser 1 5 10
<210> SEQ ID NO 139 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 139
Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp Gln Pro Glu Ala 1 5
10 15 Leu Asp Ile <210> SEQ ID NO 140 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 140 Ser Gly Asn Ile Ala Ala Asn Tyr 1 5
<210> SEQ ID NO 141 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141
Ser Ser Asp Val Gly Gly Tyr Asn Ser 1 5 <210> SEQ ID NO 142
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 142 Ser Asn Asn Val Gly Asn Gln
Gly 1 5 <210> SEQ ID NO 143 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
143 Ser Asn Asn Val Gly His Gln Gly 1 5 <210> SEQ ID NO 144
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 144 Asn Ile Gly Gly Tyr Ser 1 5
<210> SEQ ID NO 145 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 145
Gln Ser Ser Val Ser Ser Tyr 1 5 <210> SEQ ID NO 146
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 146 Gln Ser Leu Ser Ser Lys Tyr
1 5 <210> SEQ ID NO 147 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
147 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> SEQ ID NO 148
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 148 Gln Ser Ile Ser Ser Tyr 1 5
<210> SEQ ID NO 149 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 149
Thr Ser Asn Ile Gly Arg Asn His 1 5 <210> SEQ ID NO 150
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 150 Glu Asp Asp 1 <210>
SEQ ID NO 151 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 151 Glu
Val Thr 1 <210> SEQ ID NO 152 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 152 Arg Asn Asn 1 <210> SEQ ID NO 153
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 153 Arg Asn Gly 1 <210>
SEQ ID NO 154 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 154 Asp
Asp Lys 1 <210> SEQ ID NO 155 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 155 Asp Ala Ser 1 <210> SEQ ID NO 156
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 156 Gly Ala Ser 1 <210>
SEQ ID NO 157 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 157 Ser
Asn Asn 1 <210> SEQ ID NO 158 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 158 Ala Ala Ser 1 <210> SEQ ID NO 159
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 159 Ser Asn Glu 1 <210>
SEQ ID NO 160 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 160 Gln
Thr Tyr Asp Thr Asn Asn His Ala Val 1 5 10 <210> SEQ ID NO
161 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 161 Cys Ser Tyr Ala
Gly His Ser Ala Tyr Val 1 5 10 <210> SEQ ID NO 162
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 162 Ser Thr Trp Asp Ser Ser Leu
Ser Ala Val Val 1 5 10 <210> SEQ ID NO 163 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 163 Ser Val Trp Asp Ser Ser Leu Ser Ala Trp
Val 1 5 10 <210> SEQ ID NO 164 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 164 Gln Val Trp Asp Ser Gly Asn Asp Arg Pro
Leu 1 5 10 <210> SEQ ID NO 165 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 165 Gln Gln Tyr Gly Ser Ser Pro Gln 1 5
<210> SEQ ID NO 166 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 166
Gln Gln Tyr Asp Gly Val Pro Arg Thr 1 5 <210> SEQ ID NO 167
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 167 Gln Ser Tyr Asp Ser Arg Leu
Ser Ala Ser Leu 1 5 10 <210> SEQ ID NO 168 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 168 Gln Gln Tyr Asp Ser Ser Pro Tyr Thr 1 5
<210> SEQ ID NO 169 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 169
Ala Ser Trp Asp Asp Asn Leu Ser Gly Trp Val 1 5 10 <210> SEQ
ID NO 170 <211> LENGTH: 2 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 170 Ala Arg 1
<210> SEQ ID NO 171 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 171
Gln Val Gln Leu Val Gln Gly Ala Glu Val 1 5 10 <210> SEQ ID
NO 172 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 172 Val Thr Val Ser
Ser 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 172
<210> SEQ ID NO 1 <211> LENGTH: 369 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60 tcctgcaagg cttctggagg caccttcagt gacaatgcta tcagctgggt
gcgacaggcc 120 ccaggacaag ggcttgagtg gatggggggc atcattccta
tctttggaaa accaaactac 180 gcacagaagt tccagggcag agtcacgatt
actgcggacg aatccacgag cacagcctac 240 atggacctga ggagcctgag
atctgaggac acggccgttt attactgtgc gagagattca 300 gacgcgtatt
actatggttc ggggggtatg gacgtctggg gccaaggcac cctggtcacc 360
gtctcctca 369 <210> SEQ ID NO 2 <211> LENGTH: 123
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Asp Asn 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Ser Asp Ala Tyr Tyr Tyr Gly Ser Gly Gly Met Asp Val
100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 3 <211> LENGTH: 335 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
ctgcctgtgc tgactcaatc atcctctgcc tctgcttccc tgggatcctc ggtcaagctc
60 acctgcactc tgagcagtgg gcatagtaac tacatcatcg catggcatca
acagcagcca 120 gggaaggccc ctcggtactt gatgaaggtt aatagtgatg
gcagccacac caagggggac 180 gggatccctg atcgcttctc aggctccagc
tctggggctg accgctacct caccatctcc 240 aacctccagt ctgaggatga
ggctagttat ttctgtgaga cctgggacac taagattcat 300 gtcttcggaa
ctgggaccaa ggtctccgtc ctcag 335 <210> SEQ ID NO 4 <211>
LENGTH: 111 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 4 Leu Pro Val Leu Thr Gln Ser Ser Ser
Ala Ser Ala Ser Leu Gly Ser 1 5 10 15 Ser Val Lys Leu Thr Cys Thr
Leu Ser Ser Gly His Ser Asn Tyr Ile 20 25 30 Ile Ala Trp His Gln
Gln Gln Pro Gly Lys Ala Pro Arg Tyr Leu Met 35 40 45 Lys Val Asn
Ser Asp Gly Ser His Thr Lys Gly Asp Gly Ile Pro Asp 50 55 60 Arg
Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr Ile Ser 65 70
75 80 Asn Leu Gln Ser Glu Asp Glu Ala Ser Tyr Phe Cys Glu Thr Trp
Asp 85 90 95 Thr Lys Ile His Val Phe Gly Thr Gly Thr Lys Val Ser
Val Leu 100 105 110 <210> SEQ ID NO 5 <211> LENGTH: 363
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg ctcctggagg tatcttcaac
accaatgctt tcagctgggt ccgacaggcc 120 cctggacaag gtcttgagtg
ggtgggaggg gtcatccctt tgtttcgaac agcaagctac 180 gcacagaacg
tccagggcag agtcaccatt accgcggacg aatccacgaa cacagcctac 240
atggagctta ccagcctgag atctgcggac acggccgtgt attactgtgc gagaagtagt
300 ggttaccatt ttaggagtca ctttgactcc tggggcctgg gaaccctggt
caccgtctcc 360 tca 363 <210> SEQ ID NO 6 <211> LENGTH:
121 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Pro
Gly Gly Ile Phe Asn Thr Asn 20 25 30 Ala Phe Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Gly Val Ile Pro
Leu Phe Arg Thr Ala Ser Tyr Ala Gln Asn Val 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Thr Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Ser Gly Tyr His Phe Arg Ser His Phe Asp Ser Trp Gly
100 105 110 Leu Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 7 <211> LENGTH: 363 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7
caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60 tcctgcaagg ctcctggagg tatcttcaac accaatgctt tcagctgggt
ccgacaggcc 120 cctggacaag gtcttgagtg ggtgggaggg gtcatccctt
tgtttcgaac agcaagctac 180 gcacagaacg tccagggcag agtcaccatt
accgcggacg aatccacgaa cacagcctac 240 atggagctta ccagcctgag
atctgcggac acggccgtgt attactgtgc gagaagtagt 300 ggttaccatt
ttaggagtca ctttgactcc tggggcctgg gaaccctggt caccgtctcc 360 tca 363
<210> SEQ ID NO 8 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Asn
Phe Met Leu Thr Gln Pro His Ser Val Ser Ala Ser Pro Gly Lys 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Asn Ile Ala Ala Asn
20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr
Thr Val 35 40 45 Ile Tyr Glu Asp Asp Arg Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala
Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp
Tyr Tyr Cys Gln Thr Tyr Asp Thr 85 90 95 Asn Asn His Ala Val Phe
Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 9 <211> LENGTH: 333 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 9 aattttatgc
tgactcagcc ccactctgtg tcggcgtctc cggggaagac ggtgaccatc 60
tcctgcaccg gcagcagtgg caacattgcc gccaactatg tgcagtggta ccaacaacgt
120 ccgggcagtg cccccactac tgtgatctat gaggatgacc gaagaccctc
tggggtccct 180 gatcggttct ctggctccat cgacaggtcc tccaactctg
cctccctcac catctcagga 240 ctgaagactg aggacgaggc tgactactac
tgtcagactt atgataccaa caatcatgct 300 gtgttcggag gaggcaccca
cctgaccgtc ctc 333 <210> SEQ ID NO 10 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala
Ser Gly Lys His Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Gly Thr Ser Asn Ile Gly Arg Asn 20 25 30 His Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Ser Asn Glu Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Val Ser Gly Leu
Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp
Asp Asn Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 110 <210> SEQ ID NO 11 <211>
LENGTH: 330 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 11 tcctatgagc tgactcagcc accctcagcg
tctgggaaac acgggcagag ggtcaccatc 60 tcttgttctg gaggcacctc
caacatcgga cgtaatcatg ttaactggta ccagcaactc 120 ccaggaacgg
cccccaaact cctcatctat agtaatgaac agcggccctc aggggtccct 180
gaccgattct ctggctccaa atctggcacc tccgcctccc tggccgtgag tgggctccag
240 tctgaggatg aggctgatta ttactgtgca tcatgggatg acaacttgag
tggttgggtg 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> SEQ
ID NO 12 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 12 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Tyr 20 25 30 Ala
Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Thr Gly Met Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Tyr Tyr Tyr Glu Ser Ser
Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 13 <211> LENGTH: 361
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg
caccttcagc gcttatgctt tcacctgggt gcggcaggcc 120 cctggacaag
ggcttgagtg gatgggaggc atcaccggaa tgtttggcac agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aactcacgag cacagcctac
240 atggagttga gctccctgac atctgaagac acggcccttt attattgtgc
gagaggattg 300 tattactatg agagtagtct tgactattgg ggccagggaa
ccctggtcac cgtctcctca 360 g 361 <210> SEQ ID NO 14
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 14 Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Cys Ser Tyr Ala
Gly His 85 90 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
Val Leu 100 105 110 <210> SEQ ID NO 15 <211> LENGTH:
361 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 15 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaggg cttctggagg
caccttcagc gcttatgctt tcacctgggt gcggcaggcc 120 cctggacaag
ggcttgagtg gatgggaggc atcaccggaa tgtttggcac agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aactcacgag cacagcctac
240 atggagttga gctccctgac atctgaagac acggcccttt attattgtgc
gagaggattg 300 tattactatg agagtagtct tgactattgg ggccagggaa
ccctggtcac cgtctcctca 360 g 361 <210> SEQ ID NO 16
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 16 Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Lys 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Ser Cys Gln Gln Tyr Asp Gly
Val Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Thr Val Glu Ile Lys
100 105 <210> SEQ ID NO 17 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 17 cagtctgtgc tgactcagcc accctccgcg
tccgggtctc ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcag
tgacgttggt ggttataact ctgtctcctg gtaccaacag 120 cacccaggca
aagcccccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtc 180
cctgatcgct tctctgcctc caagtctggc aacacggcct ccctgaccgt ctctgggctc
240 caggctgagg atgaggctga ttatttctgc tgctcatatg caggccacag
tgcttatgtc 300 ttcggaactg ggaccaaggt caccgtcctg 330 <210> SEQ
ID NO 18 <211> LENGTH: 124 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Thr Ser Ser Glu Val Thr Phe Ser Ser Phe 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40
45 Gly Gly Ile Ser Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Gln Ser Thr Arg Thr
Ala Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Ser Tyr Ile Cys Ser Gly
Gly Thr Cys Val Phe Asp 100 105 110 His Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 <210> SEQ ID NO 19 <211>
LENGTH: 324 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 19 gaaattgtgc tgactcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca
gagtcttagc agcaagtact tagcctggta tcagcagaaa 120 cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcaccc tcaccatcag tagactggag
240 cctgaagatt ttgcagtgta ttcctgtcag cagtatgatg gcgtacctcg
gacgttcggc 300 caagggacca cggtggaaat caaa 324 <210> SEQ ID NO
20 <211> LENGTH: 110 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 20 Gln Pro Gly Leu Thr
Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr
Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30
Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35
40 45 Ser Tyr Arg Asn Asn Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe
Ser 50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
Gly Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Thr
Trp Asp Ser Ser Leu 85 90 95 Ser Ala Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO 21
<211> LENGTH: 372 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 21 caggtgcagc tggtgcagtc
aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcacgt
cctctgaagt caccttcagt agttttgcta tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gctgggaggg atcagcccta tgtttggaac acctaattac
180 gcgcagaagt tccaaggcag agtcaccatt accgcggacc agtccacgag
gacagcctac 240 atggacctga ggagcctgag atctgaggac acggccgtgt
attattgtgc gagatctcct 300 tcttacattt gttctggtgg aacctgcgtc
tttgaccatt ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210>
SEQ ID NO 22 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Asp Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 <210> SEQ ID NO 23 <211>
LENGTH: 372 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 23 caggtacagc tgcagcagtc aggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcacgt cctctgaagt
caccttcagt agttttgcta tcagctgggt gcgacaggcc 120 cctggacaag
ggcttgagtg gctgggaggg atcagcccta tgtttggaac acctaattac 180
gcgcagaagt tccaaggcag agtcaccatt accgcggacc agtccacgag gacagcctac
240 atggacctga ggagcctgag atctgaggac acggccgtgt attattgtgc
gagatctcct 300 tcttacattt gttctggtgg aacctgcgtc tttgaccatt
ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> SEQ ID NO
24 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 24 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Val Thr Phe Ser Ser Tyr 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ile Gly Val Phe Gly Val Pro Lys Tyr Ala Gln Asn Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Pro Thr Ser Thr
Val Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Gly Tyr Tyr Val Gly Lys
Asn Gly Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 25 <211> LENGTH: 330
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 cagcctgggc tgactcagcc accctcggtg
tccaagggct tgagacagac cgccacactc 60 acctgcactg ggaacagcaa
caatgttggc aaccaaggag cagcttggct gcagcagcac 120 cagggccacc
ctcccaaact cctatcctac aggaataatg accggccctc agggatctca 180
gagagattct ctgcatccag gtcaggaaac acagcctccc tgaccattac tggactccag
240 cctgaggacg aggctgacta ttactgctca acatgggaca gcagcctcag
tgctgtggta 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> SEQ
ID NO 26 <211> LENGTH: 110 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 26 Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Ile
Leu Thr Cys Thr Gly Asp Ser Asn Asn Val Gly His Gln 20 25 30 Gly
Thr Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40
45 Ser Tyr Arg Asn Gly Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser
50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ile Gly
Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp
Asp Ser Ser Leu 85 90 95 Ser Ala Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 <210> SEQ ID NO 27 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 27 gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca
gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc
ctaagctcct gatctatgct gcatccagtt tgcaaagagg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagac ttcactctca ccattagcag cctgcagcct
240 gaagattttg cagtgtatta ctgtcagcag tatgatagtt caccgtacac
ttttggccag 300 gggaccaagg tagagatcaa a 321 <210> SEQ ID NO 28
<211> LENGTH: 126 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 28 Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Arg Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Val Trp Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr
65 70 75 80 Met Glu Val Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp
Gln Pro Glu Ala 100 105 110 Leu Asp Ile Trp Gly Leu Gly Thr Thr Val
Thr Val Ser Ser 115 120 125 <210> SEQ ID NO 29 <211>
LENGTH: 366 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 29 caggtgcagc tggtgcaatc tggggctgaa
gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaaga cttctggagt
caccttcagc agctatgcta tcagttgggt gcgacaggcc 120 cctggacaag
ggcttgagtg gatgggaggg atcatcggtg tctttggtgt accaaagtac 180
gcgcagaact tccagggcag agtcacaatt accgcggaca aaccgacgag tacagtctac
240 atggagctga acagcctgag agctgaggac acggccgtgt attactgtgc
gagagagccc 300 gggtactacg taggaaagaa tggttttgat gtctggggcc
aagggacaat ggtcaccgtc 360 tcttca 366 <210> SEQ ID NO 30
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 30
Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5
10 15 Thr Ala Ser Ile Pro Cys Gly Gly Asn Asn Ile Gly Gly Tyr Ser
Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Leu Leu
Val Ile Tyr 35 40 45 Asp Asp Lys Asp Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ala 50 55 60 Asn Ser Gly Ser Thr Ala Thr Leu Thr
Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Gly Asp Tyr Tyr Cys
Gln Val Trp Asp Ser Gly Asn Asp Arg 85 90 95 Pro Leu Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 <210> SEQ ID NO 31
<211> LENGTH: 330 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 31 tcctatgagc tgactcagcc
accctcggtg tccaagggct tgagacagac cgccatactc 60 acctgcactg
gagacagcaa caatgttggc caccaaggta cagcttggct gcaacaacac 120
cagggccacc ctcccaaact cctatcctac aggaatggca accggccctc agggatctca
180 gagagattct ctgcatccag gtcaggaaat acagcctccc tgaccattat
tggactccag 240 cctgaggacg aggctgacta ctactgctca gtatgggaca
gcagcctcag tgcctgggtg 300 ttcggcggag ggaccaagct gaccgtccta 330
<210> SEQ ID NO 32 <211> LENGTH: 123 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Pro Phe Ser Met Thr
20 25 30 Ala Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys Tyr
Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Asn Thr Ala Asn 65 70 75 80 Met Glu Leu Thr Ser Leu Lys Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Leu Ser Ser
Tyr Gln Pro Asn Asn Asp Ala Phe Ala Ile 100 105 110 Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 33
<211> LENGTH: 378 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 33 caggtgcagc tggtgcagtc
tggggctgag gtgaggaagc ctggggcctc agtgaaggtc 60 tcatgtaagg
cttctggata caccttcacc ggttattata ttcactgggt gcgacaggcc 120
cctggacaag gacttgagtg gatgggttgg atcaacccta tgactggtgg cacaaactat
180 gcacagaagt ttcaggtctg ggtcaccatg acccgggaca cgtccatcaa
cacagcctac 240 atggaggtga gcaggctgac atctgacgac acggccgtgt
attactgtgc gaggggggct 300 tccgtattac gatattttga ctggcagccc
gaggctcttg atatctgggg cctcgggacc 360 acggtcaccg tctcctca 378
<210> SEQ ID NO 34 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 34 Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys
Gln Gln Tyr Gly Ser Ser Pro Gln 85 90 95 Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 105 <210> SEQ ID NO 35 <211>
LENGTH: 324 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 35 cagcctgtgc tgactcagcc accctcggtg
tcagtggccc caggacagac ggccagcatt 60 ccctgtgggg ggaacaacat
tggaggctac agtgtacact ggtaccaaca aaagccgggc 120 caggcccccc
tcttggtcat ttatgacgat aaagaccggc cctcagggat ccctgagcga 180
ttctctggcg ccaactctgg gagcacggcc accctgacaa tcagcagggt cgaagccggg
240 gatgagggcg actactactg tcaggtgtgg gatagtggta atgatcgtcc
gctgttcggc 300 ggagggacca agctgaccgt ccta 324 <210> SEQ ID NO
36 <211> LENGTH: 123 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 36 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Pro Phe Ser Met Thr 20 25 30 Ala
Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr
Ala Asn 65 70 75 80 Met Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn
Asn Asp Ala Phe Ala Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 37 <211> LENGTH:
369 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37 caggtgcagc tggtgcagtc tggggctgaa
gtgaagaagc ctggctcctc ggtgaaggtt 60 tcctgcaagg cttctggagg
ccccttcagc atgactgctt tcacctggct gcgacaggcc 120 cctggacaag
ggcttgagtg gatgggtggg atcagcccta tctttcgtac accgaagtac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagccaac
240 atggagctga ccagcctgaa atctgaggac acggccgtgt attactgtgc
gagaaccctt 300 tcctcctacc aaccgaataa tgatgctttt gctatctggg
gccaagggac aatggtcacc 360 gtctcttca 369 <210> SEQ ID NO 38
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 38 Leu Pro Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Arg
65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
Arg Leu 85 90 95 Ser Ala Ser Leu Phe Gly Thr Gly Thr Thr Val Thr
Val Leu 100 105 110 <210> SEQ ID NO 39 <211> LENGTH:
318 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 39 gaaattgtgt tgacgcagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct
240 gaagattttg cagtctattt ctgtcagcag tatggtagct cacctcaatt
cggccaaggg 300 acacgactgg agattaaa 318 <210> SEQ ID NO 40
<211> LENGTH: 369
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 40 caggtgcagc tggtgcagtc tggggctgaa
gtgaagaagc ctggctcctc ggtgaaggtt 60 tcctgcaagg cttctggagg
ccccttcagc atgactgctt tcacctggct gcgacaggcc 120 cctggacaag
ggcttgagtg gatgggtggg atcagcccta tctttcgtac accgaagtac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagccaac
240 atggagctga ccagcctgaa atctgaggac acggccgtgt attactgtgc
gagaaccctt 300 tcctcctacc aaccgaataa tgatgctttt gctatctggg
gccaagggac aatggtcacc 360 gtctcttca 369 <210> SEQ ID NO 41
<400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <211>
LENGTH: 330 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 42 ctgcctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc
caacatcgga agtaatactg taaactggta ccagcagctc 120 ccaggaacgg
cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180
gaccgattct ctggctccag gtcaggcacc tcagcctccc tggccatcat tggactccgg
240 cctgaggatg aagctgatta ttactgtcag tcgtatgaca gcaggctcag
tgcttctctc 300 ttcggaactg ggaccacggt caccgtcctc 330 <210> SEQ
ID NO 43 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus Sequence <400> SEQUENCE: 43 Ser
Tyr Ala Phe Ser 1 5 <210> SEQ ID NO 44 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 44 Thr Asn Ala Phe Ser 1 5 <210> SEQ ID
NO 45 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 45 Ala Tyr Ala Phe Thr
1 5 <210> SEQ ID NO 46 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
46 Ser Phe Ala Ile Ser 1 5 <210> SEQ ID NO 47 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 47 Ser Tyr Ala Ile Ser 1 5 <210> SEQ ID
NO 48 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 48 Gly Tyr Tyr Ile His
1 5 <210> SEQ ID NO 49 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
49 Met Thr Ala Phe Thr 1 5 <210> SEQ ID NO 50 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 50 Asp Asn Ala Ile Ser 1 5 <210> SEQ ID
NO 51 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus Sequence <400> SEQUENCE: 51 Gly
Ile Ile Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10
15 Gly <210> SEQ ID NO 52 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
52 Gly Val Ile Pro Leu Phe Arg Thr Ala Ser Tyr Ala Gln Asn Val Gln
1 5 10 15 Gly <210> SEQ ID NO 53 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 53 Gly Ile Ile Gly Met Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> SEQ ID NO 54
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 54 Gly Ile Ser Pro Met Phe Gly
Thr Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> SEQ
ID NO 55 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 55 Gly Ile Ile Gly Val
Phe Gly Val Pro Lys Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
<210> SEQ ID NO 56 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 56 Trp
Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10
15 Val <210> SEQ ID NO 57 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
57 Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys Tyr Ala Gln Lys Phe Gln
1 5 10 15 Gly <210> SEQ ID NO 58 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 58 Gly Ile Ile Pro Ile Phe Gly Lys Pro Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> SEQ ID NO 59
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Consensus Sequence <400> SEQUENCE: 59
Ser Ser Gly Tyr Tyr Tyr Gly Gly Gly Phe Asp Val 1 5 10 <210>
SEQ ID NO 60 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 60 Ser Ser
Gly Tyr His Phe Gly Arg Ser His Phe Asp Ser 1 5 10 <210> SEQ
ID NO 61 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 61 Gly Leu Tyr Tyr Tyr
Glu Ser Ser Leu Asp Tyr 1 5 10 <210> SEQ ID NO 62 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 62 Ser Pro Ser Tyr Ile Cys Ser Gly Gly Thr
Cys Val Phe Asp His 1 5 10 15 <210> SEQ ID NO 63 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 63 Glu Pro Gly Tyr Tyr Val Gly Lys Asn Gly
Phe Asp Val 1 5 10 <210> SEQ ID NO 64 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 64 Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp
Gln Pro Glu Ala Leu Asp 1 5 10 15 Ile <210> SEQ ID NO 65
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 65 Thr Leu Ser Ser Tyr Gln Pro
Asn Asn Asp Ala Phe Ala Ile 1 5 10 <210> SEQ ID NO 66
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 66 Asp Ser Asp Ala Tyr Tyr Tyr
Gly Ser Gly Gly Met Asp Val 1 5 10 <210> SEQ ID NO 67
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 67 Thr Gly Ser Ser Ser Asn Ile
Gly Asn Tyr Val Ala 1 5 10 <210> SEQ ID NO 68 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 68 Thr Gly Ser Ser Ser Asn Ile Ala Ala Asn
Tyr Val Gln 1 5 10 <210> SEQ ID NO 69 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
Sequence <400> SEQUENCE: 69 Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr Asn Ser Val Ser 1 5 10 <210> SEQ ID NO 70 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 70 Thr Gly Asn Ser Asn Asn Val Gly Asn Gln
Gly Ala Ala 1 5 10 <210> SEQ ID NO 71 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 71 Thr Gly Asp Ser Asn Asn Val Gly His Gln
Gly Thr Ala 1 5 10 <210> SEQ ID NO 72 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 Gly Gly Asn Asn Ile Gly Gly Tyr Ser Val
His 1 5 10 <210> SEQ ID NO 73 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 73 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
Ala 1 5 10 <210> SEQ ID NO 74 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Arg Ala Ser Gln Ser Leu Ser Ser Lys Tyr
Leu Ala 1 5 10 <210> SEQ ID NO 75 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 75 Thr Gly Ser Ser Ser Asn Ile Gly Asn Tyr
Val Ala 1 5 10 <210> SEQ ID NO 76 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 76 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
Thr Val Asn 1 5 10 <210> SEQ ID NO 77 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 77 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu
Asn 1 5 10 <210> SEQ ID NO 78 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 78 Thr Leu Ser Ser Gly His Ser Asn Tyr Ile
Ile Ala 1 5 10 <210> SEQ ID NO 79 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
Sequence <400> SEQUENCE: 79 Ser Asn Ser Asp Arg Pro Ser 1 5
<210> SEQ ID NO 80 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 80 Glu
Asp Asp Arg Arg Pro Ser 1 5 <210> SEQ ID NO 81 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Glu Val Thr Lys Arg Pro Ser 1 5
<210> SEQ ID NO 82 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 Arg
Asn Asn Asp Arg Pro Ser 1 5 <210> SEQ ID NO 83 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 83 Arg Asn Gly Asn Arg Pro Ser 1 5
<210> SEQ ID NO 84 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 84 Asp
Asp Lys Asp Arg Pro Ser 1 5 <210> SEQ ID NO 85 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 85 Asp Ala Ser Asn Arg Ala Thr 1 5
<210> SEQ ID NO 86 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 86 Gly
Ala Ser Ser Arg Ala Thr 1 5 <210> SEQ ID NO 87 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Ser Asn Asn Gln Arg Pro Ser 1 5
<210> SEQ ID NO 88 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 88 Ala
Ala Ser Ser Leu Gln Arg 1 5 <210> SEQ ID NO 89 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 Ser Asn Glu Gln Arg Pro Ser 1 5
<210> SEQ ID NO 90 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 Val
Asn Ser Asp Gly Ser His Thr Lys Gly Asp 1 5 10 <210> SEQ ID
NO 91 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus Sequence <400> SEQUENCE: 91 Gln
Ser Tyr Asp Ser Leu Ser Ala Tyr Val 1 5 10 <210> SEQ ID NO 92
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 92 Gln Ser Tyr Asp Thr Asn Asn
His Ala Val 1 5 10 <210> SEQ ID NO 93 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Cys Ser Tyr Ala Gly His Ser Ala Tyr Val 1
5 10 <210> SEQ ID NO 94 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
94 Ser Thr Trp Asp Ser Ser Leu Ser Ala Val Val 1 5 10 <210>
SEQ ID NO 95 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 95 Ser Val
Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> SEQ ID NO 96
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 96 Gln Val Trp Asp Ser Gly Asn
Asp Arg Pro Leu 1 5 10 <210> SEQ ID NO 97 <211> LENGTH:
9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Gln Gln Tyr Gly Ser Ser Pro Gln Val 1 5
<210> SEQ ID NO 98 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Gln
Gln Tyr Asp Gly Val Pro Arg Thr 1 5 <210> SEQ ID NO 99
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 99 Gln Ser Tyr Asp Ser Arg Leu
Ser Ala Ser Leu 1 5 10 <210> SEQ ID NO 100 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 100 Gln Gln Tyr Asp Ser Ser Pro Tyr Thr 1 5
<210> SEQ ID NO 101 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 101
Ala Ser Trp Asp Asp Asn Leu Ser Gly Trp Val 1 5 10 <210> SEQ
ID NO 102 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 102 Glu Thr Trp Asp
Thr Lys Ile His Val 1 5 <210> SEQ ID NO 103 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide <400> SEQUENCE: 103 Gly Thr Glu Thr Ser Gln
Val Ala Pro Ala 1 5 10 <210> SEQ ID NO 104 <211>
LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic 6xHis tag <400>
SEQUENCE: 104 His His His His His His 1 5 <210> SEQ ID NO 105
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Influenza A virus <400> SEQUENCE: 105 Ile Asn Gly Trp 1
<210> SEQ ID NO 106 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Influenza A virus <400> SEQUENCE:
106 Ile Asp Gly Trp 1 <210> SEQ ID NO 107 <211> LENGTH:
4 <212> TYPE: PRT <213> ORGANISM: Influenza A virus
<400> SEQUENCE: 107 Val Ala Gly Trp 1 <210> SEQ ID NO
108 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Influenza A virus <400> SEQUENCE: 108 Val Asp Gly
Trp 1 <210> SEQ ID NO 109 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Influenza A virus <400>
SEQUENCE: 109 Ile Ala Gly Trp 1 <210> SEQ ID NO 110
<211> LENGTH: 87 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 110 Lys Lys Pro Gly Ser Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Gly 1 5 10 15 Thr Phe Ser Ser Tyr
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu
Trp Met Gly Gly Ile Ile Pro Met Phe Gly Thr Pro Asn 35 40 45 Tyr
Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser 50 55
60 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg 85 <210> SEQ ID NO
111 <211> LENGTH: 87 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 111 Lys Lys Pro Gly
Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 1 5 10 15 Thr Phe
Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 20 25 30
Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Pro Asn 35
40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser 50 55 60 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg 85 <210>
SEQ ID NO 112 <211> LENGTH: 108 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 112 Lys
Lys Pro Gly Ser Ser Val Lys Val Ser Cys Thr Ser Ser Glu Val 1 5 10
15 Thr Phe Ser Ser Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
20 25 30 Gly Leu Glu Trp Leu Gly Gly Ile Ser Pro Met Phe Gly Thr
Pro Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr
Ala Asp Gln Ser 50 55 60 Thr Arg Thr Ala Tyr Met Asp Leu Arg Ser
Leu Arg Ser Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg Ser
Pro Ser Tyr Ile Cys Ser Gly Gly 85 90 95 Thr Cys Val Phe Asp His
Trp Gly Gln Gly Thr Leu 100 105 <210> SEQ ID NO 113
<211> LENGTH: 104 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 113 Lys Lys Pro Gly Ser Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Gly 1 5 10 15 Thr Phe Ser Ala Tyr
Ala Phe Thr Trp Val Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu
Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Ala Asn 35 40 45 Tyr
Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu 50 55
60 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr
65 70 75 80 Ala Leu Tyr Tyr Cys Ala Arg Gly Leu Tyr Tyr Tyr Glu Ser
Ser Phe 85 90 95 Asp Tyr Trp Gly Gln Gly Thr Leu 100 <210>
SEQ ID NO 114 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 114 Lys
Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 1 5 10
15 Pro Phe Ser Met Thr Ala Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln
20 25 30 Gly Leu Glu Trp Met Gly Gly Ile Ser Pro Ile Phe Arg Thr
Pro Lys 35 40 45 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser 50 55 60 Thr Asn Thr Ala Asn Met Glu Leu Thr Ser
Leu Lys Ser Glu Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg Thr
Leu Ser Ser Tyr Gln Pro Asn Asn 85 90 95 Asp Ala Phe Ala Ile Trp
Gly Gln Gly Thr Met 100 105 <210> SEQ ID NO 115 <211>
LENGTH: 106 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 115 Lys Lys Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Val 1 5 10 15 Thr Phe Ser Ser Tyr Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 20 25 30 Gly Leu Glu Trp
Met Gly Gly Ile Ile Gly Val Phe Gly Val Pro Lys 35 40 45 Tyr Ala
Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Pro 50 55 60
Thr Ser Thr Val Tyr Met Glu Leu Asn Ser Leu Arg Ala Glu Asp Thr 65
70 75 80 Ala Val Tyr Tyr Cys Ala Arg Glu Pro Gly Tyr Tyr Val Gly
Lys Asn 85 90 95 Gly Phe Asp Val Trp Gly Gln Gly Thr Met 100 105
<210> SEQ ID NO 116 <211> LENGTH: 105 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 116
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Pro Gly Gly 1 5
10 15 Ile Phe Asn Thr Asn Ala Phe Ser Trp Val Arg Gln Ala Pro Gly
Gln 20 25 30 Gly Leu Glu Trp Val Gly Gly Val Ile Pro Leu Phe Arg
Thr Ala Ser 35 40 45 Tyr Ala Gln Asn Val Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser 50 55 60
Thr Asn Thr Ala Tyr Met Glu Leu Thr Ser Leu Arg Ser Ala Asp Thr 65
70 75 80 Ala Val Tyr Tyr Cys Ala Arg Ser Ser Gly Tyr His Phe Arg
Ser His 85 90 95 Phe Asp Ser Trp Gly Leu Gly Thr Leu 100 105
<210> SEQ ID NO 117 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 117
Arg Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 1 5
10 15 Thr Phe Thr Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Gln 20 25 30 Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Met Thr Gly
Gly Thr Asn 35 40 45 Tyr Ala Gln Lys Phe Gln Val Trp Val Thr Met
Thr Arg Asp Thr Ser 50 55 60 Ile Asn Thr Ala Tyr Met Glu Val Thr
Arg Leu Thr Ser Asp Asp Thr 65 70 75 80 Ala Val Tyr Tyr Cys Ala Arg
Gly Ala Ser Val Leu Arg Tyr Phe Asp 85 90 95 Trp Gln Pro Glu Ala
Leu Asp Ile Trp Gly Leu Gly Thr Thr 100 105 110 <210> SEQ ID
NO 118 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 118 Gly Gly Thr Phe
Ser Ser Tyr Ala 1 5 <210> SEQ ID NO 119 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5
<210> SEQ ID NO 120 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 120
Glu Val Thr Phe Ser Ser Phe Ala 1 5 <210> SEQ ID NO 121
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 121 Gly Gly Thr Phe Ser Ala Tyr
Ala 1 5 <210> SEQ ID NO 122 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
122 Gly Gly Pro Phe Ser Met Thr Ala 1 5 <210> SEQ ID NO 123
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 123 Gly Val Thr Phe Ser Ser Tyr
Ala 1 5 <210> SEQ ID NO 124 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
124 Gly Gly Ile Phe Asn Thr Asn Ala 1 5 <210> SEQ ID NO 125
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 125 Gly Tyr Thr Phe Thr Gly Tyr
Tyr 1 5 <210> SEQ ID NO 126 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
126 Ile Ile Pro Met Phe Gly Thr Pro 1 5 <210> SEQ ID NO 127
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 127 Ile Ile Pro Ile Phe Gly Thr
Pro 1 5 <210> SEQ ID NO 128 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
128 Ile Ser Pro Met Phe Gly Thr Pro 1 5 <210> SEQ ID NO 129
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 129 Ile Ile Gly Met Phe Gly Thr
Ala 1 5 <210> SEQ ID NO 130 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
130 Ile Ser Pro Ile Phe Arg Thr Pro 1 5 <210> SEQ ID NO 131
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 131 Ile Ile Gly Val Phe Gly Val
Pro 1 5 <210> SEQ ID NO 132 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
132 Val Ile Pro Leu Phe Arg Thr Ala 1 5 <210> SEQ ID NO 133
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 133 Ile Asn Pro Met Thr Gly Gly
Thr 1 5 <210> SEQ ID NO 134 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 134 Ala Arg Ser Pro Ser Tyr Ile Cys Ser Gly
Gly Thr Cys Val Phe Asp 1 5 10 15 His <210> SEQ ID NO 135
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 135 Ala Arg Gly Leu Tyr Tyr Tyr
Glu Ser Ser Phe Asp Tyr 1 5 10 <210> SEQ ID NO 136
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 136 Ala Arg Thr Leu Ser Ser Tyr
Gln Pro Asn Asn Asp Ala Phe Ala Ile 1 5 10 15
<210> SEQ ID NO 137 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 137
Ala Arg Glu Pro Gly Tyr Tyr Val Gly Lys Asn Gly Phe Asp Val 1 5 10
15 <210> SEQ ID NO 138 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
138 Ala Arg Ser Ser Gly Tyr His Phe Arg Ser His Phe Asp Ser 1 5 10
<210> SEQ ID NO 139 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 139
Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp Gln Pro Glu Ala 1 5
10 15 Leu Asp Ile <210> SEQ ID NO 140 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 140 Ser Gly Asn Ile Ala Ala Asn Tyr 1 5
<210> SEQ ID NO 141 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141
Ser Ser Asp Val Gly Gly Tyr Asn Ser 1 5 <210> SEQ ID NO 142
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 142 Ser Asn Asn Val Gly Asn Gln
Gly 1 5 <210> SEQ ID NO 143 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
143 Ser Asn Asn Val Gly His Gln Gly 1 5 <210> SEQ ID NO 144
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 144 Asn Ile Gly Gly Tyr Ser 1 5
<210> SEQ ID NO 145 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 145
Gln Ser Ser Val Ser Ser Tyr 1 5 <210> SEQ ID NO 146
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 146 Gln Ser Leu Ser Ser Lys Tyr
1 5 <210> SEQ ID NO 147 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
147 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> SEQ ID NO 148
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 148 Gln Ser Ile Ser Ser Tyr 1 5
<210> SEQ ID NO 149 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 149
Thr Ser Asn Ile Gly Arg Asn His 1 5 <210> SEQ ID NO 150
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 150 Glu Asp Asp 1 <210>
SEQ ID NO 151 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 151 Glu
Val Thr 1 <210> SEQ ID NO 152 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 152 Arg Asn Asn 1 <210> SEQ ID NO 153
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 153 Arg Asn Gly 1 <210>
SEQ ID NO 154 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 154 Asp
Asp Lys 1 <210> SEQ ID NO 155 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 155 Asp Ala Ser 1 <210> SEQ ID NO 156
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 156 Gly Ala Ser 1 <210>
SEQ ID NO 157 <211> LENGTH: 3 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 157 Ser
Asn Asn 1 <210> SEQ ID NO 158 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 158 Ala Ala Ser 1 <210> SEQ ID NO 159
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 159
Ser Asn Glu 1 <210> SEQ ID NO 160 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 160 Gln Thr Tyr Asp Thr Asn Asn His Ala Val 1
5 10 <210> SEQ ID NO 161 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
161 Cys Ser Tyr Ala Gly His Ser Ala Tyr Val 1 5 10 <210> SEQ
ID NO 162 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 162 Ser Thr Trp Asp
Ser Ser Leu Ser Ala Val Val 1 5 10 <210> SEQ ID NO 163
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 163 Ser Val Trp Asp Ser Ser Leu
Ser Ala Trp Val 1 5 10 <210> SEQ ID NO 164 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 164 Gln Val Trp Asp Ser Gly Asn Asp Arg Pro
Leu 1 5 10 <210> SEQ ID NO 165 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 165 Gln Gln Tyr Gly Ser Ser Pro Gln 1 5
<210> SEQ ID NO 166 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 166
Gln Gln Tyr Asp Gly Val Pro Arg Thr 1 5 <210> SEQ ID NO 167
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 167 Gln Ser Tyr Asp Ser Arg Leu
Ser Ala Ser Leu 1 5 10 <210> SEQ ID NO 168 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 168 Gln Gln Tyr Asp Ser Ser Pro Tyr Thr 1 5
<210> SEQ ID NO 169 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 169
Ala Ser Trp Asp Asp Asn Leu Ser Gly Trp Val 1 5 10 <210> SEQ
ID NO 170 <211> LENGTH: 2 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 170 Ala Arg 1
<210> SEQ ID NO 171 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 171
Gln Val Gln Leu Val Gln Gly Ala Glu Val 1 5 10 <210> SEQ ID
NO 172 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 172 Val Thr Val Ser
Ser 1 5
* * * * *
References